CN106574244B - Expansion of lymphocytes with cytokine compositions for active cellular immunotherapy - Google Patents

Abstract

The present invention relates to a composition for expanding lymphocytes comprising at least two selected from the group consisting of interleukin 2 (IL-2), interleukin 15 (IL-15) and interleukin 21 (IL-21). types of cytokines. The present invention also relates to a method for preparing a clinically relevant lymphocyte population, comprising the steps of: obtaining a body sample, in particular a tissue sample or a body fluid sample, comprising at least one lymphocyte from a mammal and selectively isolating cells in the body sample; in vitro Culturing a body sample to expand and/or stimulate lymphocytes in the sample, wherein the culturing includes the use of IL-2, IL-15 and/or IL-21; and selectively determining the presence of clinically relevant lymphocytes in the cultured sample exist. The present invention also relates to immunotherapy and clinically relevant lymphocyte populations.

Description

Expansion of lymphocytes with cytokine compositions for active cellular immunotherapy

Technical Field

The present invention relates to active cellular immunotherapy comprising a method of preparing a clinically relevant lymphocyte population using a composition of predetermined cytokines. The invention also relates to cytokine compositions and clinically relevant lymphocytes produced.

Background

Cancer remains one of the most common causes of death in developed countries. For example, in the united states and germany, it is the second most common cause of death, with mortality numbers 560,000(2009) and 218,000(2010), respectively. Despite the improved ability to detect and treat this range of diseases, the survival rates for many cancers remain low.

Among the cancer diseases, pancreatic cancer is the fourth leading cause of cancer death in the united states and sweden, and there are no signs of improvement. By the time pancreatic cancer is diagnosed, most patients with locally advanced cancer or metastatic disease are not cured and only allowed palliative treatment. Median survival was about 6 months. Only about 15 to 20% of patients have resectable tumors and thus the disease is likely to be cured. Like most cancers, pancreatic cancer is a systemic disease requiring early and systemic intervention. Pancreatic cancer is highly resistant to chemotherapy and radiation, as compared to many other types of cancer. In order to reach a significant breakthrough in improving the poor prognosis of pancreatic cancer, it is imperative to find new alternatives and more effective therapeutic concepts. The biology of pancreatic cancer is associated with local and systemic immunosuppression that allows tumor progression and metastasis.

The situation is similar in glioblastoma, the most common progressive glioma, with an incidence of 2-3/100.000 in the united states. Glioblastoma accounts for 12 to 15% of all intracranial tumors, 50 to 60% of histiocytic tumors. The new treatment regimen increased median overall survival (14.6 months for radiation plus temozolomide compared to 12.1 months for radiation alone). To date, the desire to develop potent and clinically effective immunotherapeutic regimens has been frustrated for patients with glioblastoma or patients with pancreatic cancer. One approach to treating these cancers is to overcome tumor-induced suppression and/or induce anti-tumor directed cellular and humoral immune responses.

One of the most promising advances is a new class of therapeutic approaches known as Active Cellular Immunotherapy (ACI). Cancer immunotherapy can be passive or active. Passive therapy is based on adoptive transfer of immune modulators including cytokines, tumor-specific antibodies or immune cells. These substances or cells are then administered to the patient to elicit an anti-tumor effect. Generally, these treatments do not produce immunological memory and therefore require chronic infusion-based treatments. Active immunotherapy, on the other hand, stimulates the patient's immune system with the aim of using the body's own immune cells to promote antigen-specific anti-tumor effects. In addition, active immunotherapy attempts to generate a durable anti-tumor response that can protect the body from minimal residual disease and tumor recurrence.

Clinically relevant long-term remission using T cells directed against tumors (tumor-reactive T cells) has been achieved in patients with melanoma (2, 3). A recent milestone paper suggests that optimal and long-lasting responses in cancer therapy are obtained if the patient's own T cells are directed against the patient's own tumor cells, i.e. the patient's own "private" mutations (4). This promising result was also obtained by adoptive transfer of T cells targeting mutant epitopes in epithelial cancers for patients with epithelial tumors (5). These methods typically rely on harvesting Tumor Infiltrating Lymphocytes (TILs) from tumor lesions or T cells from peripheral blood.

A recent report by the CTEP adoptive cell therapy team committee summarizes the protocol for expanding tumor-reactive T cells and TILs from peripheral blood.

This study compiles a roadmap for using TIL therapy or T cell-based therapy, with particular attention to product consistency and efficient yield of T cell products. The consistency and yield of anti-melanoma directed T cells appears to be achievable with current methodologies that enable T cell-based strategies to be taken into mainstream cancer therapy with biologies, i.e., anti-CD 40L or PD-1 targeted therapies.

TIL in mild culture (minimally cultured) appears to provide the most effective phenotype and properties (profile) for clinical use (11). The most successful approach to date has been to use autologous ex vivo (ex vivo) activated T cells grown in 24-well plates, to test immune effector function and to perform further expansion using IL-2, allogeneic feeder cells and OKT3 (9, 12, 13).

CD4+ or CD8+ tumor antigen (TAA) -directed T cells have been generated from peripheral blood under GMP conditions and are formulated for subsequent treatment of patients. This has been achieved with autologous CD4+ T cells (14 to 16) or CD8+ T cells (17), some T cells against the NY-ESO-1 antigen (18), which is also possible in PBMCs from healthy patients without cancer, because there are sufficient T cell precursors in the peripheral circulation. Various methods of expanding T cells that produce CD8+ T cell clones for targeted therapy have been described (19). This would be of great interest, since clonal repopulation (clonal repopulation) of the immune system of patients with anti-tumor lymphocytes has been shown to induce cancer regression but also to display autoimmunity (20).

Growth medium formulations may also be associated with successful active immunotherapy. Studies have shown that starvation affects T cell mediated immune responses and can induce starvation-induced immunosuppression, however expansion of certain T cell subsets can also be induced. This mechanism appears to be mediated by leptin (21), which also regulates B cell development and subsequent B cell responses (22). This led to the discovery of a nutrient sensor pathway (GCN 2 in dendritic cells) that enhanced antigen presentation (23). More recent studies have shown that cytokine-driven T cell expansion (such as those in ex vivo expansion of TIL or ACT) is dependent on exogenous amino acids, and that the cytokine, IL-7, up-regulates genes associated with amino acid transporter expression. Thus, the customized growth medium requirements will be influenced by the corresponding cytokine mixture used for T cell expansion (24) and the amino acids in the medium; both factors affect the maturation and differentiation of T cells, which is clinically significant.

Clinical (anti-tumor) effects appear to be mediated by CD8+ and central memory cells, defined by CD45RA-CCR7+, defined ex vivo from patients responding to T cell-based therapy. The phenotype of such T cells is determined by the ex vivo expanded T cell population and host factors following adoptive transfer. Targeting multiple T cell populations of cancer cells may be beneficial for effective immune responses, including long-term memory T cells as well as T cells that can immediately respond to (cancer) target cells and generate an anti-tumor directed immune response, including terminally differentiated T cells expressing cytolytic molecules such as granzyme and perforin (25, 26). The immunological memory of long-term memory is determined in part by the increased proliferative potential and half-life that can be measured by telomere length (27, 28).

Due to the differences in gene expression in vitro and in vivo and the different cytokine milieu in each patient, little is known what stage of the (melanoma) -specific TIL or T cell clone is most favorable for metastasis in vivo. Not only individual phenotypes, but rather different phenotypes associated with rapid delivery of immune effector functions (terminally differentiated CD45RA + CCR7-), T cells and long-term immunological memory that provides central memory T cells that can complement the differentiated T cell pool, may represent a good choice for T cell expansion.

Also relevant appears to be the expression of activation/depletion markers (i.e., LAG-3, PD-1 and/or 4-1BB) on T cells, which may indicate higher variation in T cell depletion and loss of function as well as enrichment of tumor antigen-specific T cells, which tend to be PD1+, and/or LAG-3, 4-1BB + (29).

NY-ESO-1 is said to be a qualified target for tumor antigen-specific T cells. NY-ESO-1 is a cancer detection antigen (36, 37), expressed in a large number of tumors. For example, NY-ESO-1 protein expression was screened in Karolinska for 50 glioblastoma lesions, and 35% of GB grade 3 and GB grade 4 were found to be NY-ESO-1 positive. Pancreatic cancer lesion screening shows a lower number of NY-ESO-1 protein + cancer lesions, in particular metastatic lesions, in the 20% range. Targeting NY-ESO-1 to expand tumor-reactive T cells from peripheral blood appears to be a "safe choice" for the target, as NY-ESO-1 appears to be expressed only in malignant cells and testis, with no apparent "off-target" reactivity in directed T cells against NY-ESO-1 (36). This has attracted great interest because clonal re-proliferation of the patient's immune system with anti-tumor lymphocytes has been shown to induce cancer regression and autoimmunity (20), which is potentially dangerous. NY-ESO-1 has been tested in many studies as a potential target in GB and GB stem cells (38), while using DNA methylating agents to increase the reactivity of NY-ESO-1 (39, 40).

In view of the state of the art, it is an object of the present invention to provide an improved method of immunotherapy.

Disclosure of Invention

The invention is based inter alia on the following findings: compositions comprising the cytokines interleukin-2 (IL-2), interleukin-15 (IL-15) and/or interleukin-21 (IL-21) result in excellent stimulation and expansion of lymphocytes, particularly clinically relevant lymphocytes. The expansion and stimulation process using cytokine mixtures is highly sensitive and allows the preparation of clinically relevant lymphocyte populations even at very low initial concentrations in the sample.

Thus, according to a first aspect, the present invention provides a composition for expanding lymphocytes, comprising at least two types of cytokines selected from interleukin 2(IL-2), interleukin-15 (IL-15) and interleukin 21 (IL-21).

Using this cytokine composition, the inventors were able to identify a novel method for preparing antigen-edited (antigen-edited) lymphocyte populations. Thus, according to a second aspect, the present invention provides a method of preparing a clinically relevant lymphocyte population, said method comprising the steps of:

-obtaining a body sample (body sample) comprising at least one lymphocyte, in particular a tissue sample or a body fluid sample, from a mammal and optionally isolating cells in said body sample,

-culturing said body sample in vitro to expand and/or stimulate lymphocytes in said sample, wherein said culturing comprises the use of IL-2, IL-15 and/or IL-21,

and optionally determining the presence of clinically relevant lymphocytes in the cultured sample.

The method of the second aspect of the invention results in the formation of a lymphocyte population, including a clinically relevant lymphocyte population.

According to a third aspect, the present invention provides a clinically relevant lymphocyte obtainable by a method according to the second aspect, wherein the clinically relevant lymphocyte is selected from a B cell, an NK cell and a T cell.

According to a fourth aspect, the present invention provides a population of lymphocytes obtained by the second aspect of the invention, the population of lymphocytes comprising a clinically relevant population of lymphocytes.

The clinically relevant lymphocyte population obtained with the method according to the second aspect of the invention is particularly advantageous for cellular immunotherapy.

According to a fifth aspect, the present invention provides an immunotherapy for the treatment or prevention of a neoplastic disease, an infectious disease or an autoimmune disease in a mammal, comprising the steps of: generating a population of clinically relevant lymphocytes according to the second aspect of the invention, wherein the body sample is obtained from the mammal; and administering the clinically relevant lymphocyte population to the mammal.

According to a sixth aspect, the present invention provides a composition according to the first aspect of the invention for use in medical treatment, in particular for use in the treatment and prevention of infectious diseases, autoimmune diseases or neoplastic diseases.

Thus, according to a seventh aspect, the present invention provides a kit for use in medical treatment, in particular for use in the treatment or prevention of an infectious disease, an autoimmune disease or a neoplastic disease, wherein the kit comprises IL-2, IL-15 and IL-21, and optionally at least one TCR-stimulating component, in particular OKT3, a co-stimulatory molecule, feeder cells and a peptide comprising the amino acid sequence of at least one clinically relevant antigen.

Drawings

FIG. 1 shows three graphs representing the results of flow cytometry analysis of samples from PBMC amplification with cytokine mixtures IL-2, IL-15 and IL-21 in combination with zoledronic acid. Samples were taken at different time points as shown on the graph. The measured signals are the CD3 signal in the Y-axis direction and the TCR γ δ signal in the X-axis direction. γ δ T cells were found to be present in the rectangular region. The primary color images show the intensity of the superimposed signal in gray scales. The percentage of cells in the rectangular area is shown above.

Figure 2 shows the results of flow cytometric analysis of lymphocytes from PBMCs expanded with cytokine mixtures in the presence of PRDM2 peptide. The larger panel on the left shows the results for the sample at the beginning of lymphocyte expansion, and the right panel shows the results for the sample after 18 days of stimulation. Cell signals were isolated based on the CD4/CD8 marker. The panels on the right show the gates (gating) of lymphocytes and CD3+ cells.

FIG. 3 shows a flow cytometric analysis of the same samples as in FIG. 2, with separation of the cell signals by IFN-. gamma.markers and size (side scattered light SSC).

Figure 4 shows the results of flow cytometric analysis of PBMC-expanded samples stimulated with cytokine cocktail and with INO80E and UCHL 3. Cell signals were gated with lymphocytes (4a), CD3+ (4b) and then isolated based on CD8 and CD4 signals (fig. 4 c).

Figure 5 shows IFN- γ production on the double negative and CD8+ populations following stimulation with INO80E or UCHL 3.

Figure 6 shows an analysis of PBMC cells expanded with cytokine cocktail and INO80E and UCHL3 peptides. Cells stimulated with INO80E were analyzed for production of cytokines CD107a (6d), CD127(6e) and CD117(6 f).

Figures 7a to 7f show the results of PBMC expansion stimulated with cytokine mixtures and with CMVpp 65.

FIG. 8 shows the results of an analysis of IFN-. gamma.after stimulation of expanded cells with NY-ESO-1: fig. 8a and 8c were unstimulated on days 0 and 18, respectively. FIG. 8b and FIG. 8d were stimulated with NY-ESO-1 on day 0 and day 18, respectively.

Figure 9 shows cytokine production from cells expanded from PBMCs obtained from glioblastoma patients upon re-stimulation with survivin on days 0 and 18. The cytokines measured were IL-2, IFN-. gamma.and TNF-. alpha.. Fig. 9a shows the results for CD4+ T cell subpopulation, fig. 9b for double negative T cell subpopulation, and fig. 9c for CD8+ T cell subpopulation.

Figure 10 shows the analysis of the phenotype of lymphocytes using flow cytometry, CD45RA and CCR 7. Lymphocytes were measured again on day 0 and after 18 days of expansion with cytokine cocktail.

Figure 11 shows an analysis of the effect of expansion on the phenotype of CD4+ cells (TH1/TH2) and CD8+ T cells.

Figure 12 shows an analysis of the cytokine CD107a expression by cells expanded from HPV patient peripheral blood. Figure 12a shows CD107a expression upon HPV L1 peptide stimulation. Fig. 12b shows the positive control and fig. 12c shows the results (medium) without stimulation. Gating process of CD8+ T cells is shown in FIGS. 12 d-12 f.

FIG. 13 shows two graphs showing IFN-. gamma.production by lymphocytes amplified in the absence of cytokines, IL-2, IL-15, IL-21, or IL-7 and IL-2, under stimulation by NY-ESO-1 or survivin.

FIG. 14 shows three graphs representing IFN- γ production by lymphocytes amplified in the absence of cytokines, IL-2, IL-15, IL-21, or IL-7 and IL-2, under stimulation by EBNA-1, EBNA-3a, or CMVpp 65.

FIG. 15 shows determination of T before and after expansion of T cells with cytokine mixtures_reg(regulatory T cells) identified flow cytometric analysis results. From left to right: t cells were gated with CD4+ T cells and then with CD25high (CD25high), indicating high expression of IL-2 receptor on activated T cells. Cells were then gated with Il-2R (high CD125) cells and tested for (intracellular) expression of Il-7 receptor (CD127) and Foxp 3.

FIG. 16 shows flow cytometric analysis to determine the percentage of PD-1+ T cells in the CD8+ subpopulation.

Fig. 17 shows specific cytolysis of expanded lymphocytes against autologous B cells loaded with peptides 1 to 12 (pulsed).

FIG. 18 shows flow cytometric analysis of PBMCs prior to IL-2/IL-15/IL-21 driven expansion in the presence of the tumor associated antigen NY-ESO-1. First, CD3+ T cells were gated, and then CD3+ T cells were gated with CD4+ T cells and CD8+ T cells.

FIG. 19 shows flow cytometric analysis of PBMCs prior to IL-2/IL-15/IL-21 driven expansion in the presence of the tumor associated antigen NY-ESO-1. First, CD3+ T cells were gated, and then CD3+ T cells were gated with CD4+ T cells and CD8+ T cells.

FIG. 20 shows a graph of tumor infiltrating lymphocyte cultures cultured in vitro with cytokines IL-2, IL-15, and IL-21, incubated for one week.

Figure 21 shows a functional protocol overview of the cytotoxicity assay of expanded lymphocytes on autologous tumor cells using radioactive (Cr51) labeling and release.

Fig. 22 shows the results of flow cytometric analysis of TIL-expanded lymphocytes obtained from a patient with glioblastoma. Fig. 22(a) shows the distribution of T cell phenotypes (from 16TIL to a specific phenotype) in the expanded TIL: precursor T cells (CD45RA + CCR7+), central memory T cells (CD45RA-CCR7+), peripheral memory T cells (CD45RA-CCR7-) and differentiated effector T cells (effector T-cells) (CD45RA + CCR7-), directed against basal phenotype CD8+ (middle panel), CD4+ (left panel) and double negative T cells (right panel), respectively. Each data point represents the percentage of a particular phenotype based on the underlying phenotype. This data indicates that IL-2, IL-15, and IL-21 amplify TILs with long-term memory phenotypes as well as T-cell precursors that provide long-term immune protection.

Fig. 22(B) shows expression of T cell activation markers and depletion markers. As in (a), the results were grouped according to basal phenotypes, CD8+ (middle panel), CD4+ (left panel), and double negative T cells (right panel). Each data point represents the percentage of cells expressing the indicated marker on the X-axis based on the basal phenotype. CD117(c-kit) is a "stem cell" related marker and indicates T cells with long-term memory, CD107a represents a marker of recent T cell degranulation. This data indicates that TIL expression amplified in IL-2, IL-15 and IL-21 enables their use as markers (e.g., c-kit) for long-term immune cell memory and immune monitoring.

FIG. 23 shows the results of flow cytometric analysis of lymphocytes (TILs) expanded from tumor tissues of pancreatic cancer patients. The left panel shows the distribution of CD4+ T cells into precursor T cells (CD45RA + CCR7+), central memory T cells (CD45RA-CCR7+), peripheral memory T cells (CD45RA-CCR7-), and differentiated effector T cells (CD45RA + CCR 7-). The right panel shows the distribution of CD8+ cells. This data indicates that IL-2, IL-15, and IL-21 amplify TILs with long-term memory phenotypes and T cell precursors that can provide long-term immune protection.

FIG. 24 shows the results of flow cytometric analysis of T cell activation markers and depletion markers (4-1BB, LAG-3, TIM-3, etc.) of lymphocytes (TILs) expanded from tumor tissues of pancreatic cancer patients. The results were grouped according to the CD4+/CD8+ phenotype CD4+ (upper panel), CD8+ (middle panel), DN (lower panel). Each data point represents the percentage of cells expressing the indicated marker on the X-axis based on the basal phenotype. This data indicates that TIL expression indicates a strong anti-tumor response and a broad range of markers of recent antigen exposure. The CD127 molecule (IL-7R) mediates potent T cell survival factors.

Figure 25 shows TCR length distribution of T cells amplified from tumor tissue of pancreatic cancer patients, determined by PCR-based methods.

Figure 26 shows the results of an intracellular cytokine production assay in CD4+, CD8, or DN T cells in lymphocytes expanded from glioblastoma. The graph in figure 7B shows the percentage of T cells producing the cytokines IFN γ and TNF α after stimulation. Fig. 12A shows the maximum stimulation of PMA/ionomycin (positive control) and background in medium only. FIG. 7B shows the results of stimulation with synthetic peptides derived from tumor associated antigens, i.e., EGRvrIII, NY-ESO-1 or survivin. This data indicates that IL-2, IL-15, and IL-21 expanded TILs from glioblastoma patients contain T cells that respond at a low frequency to common tumor-associated antigens.

Figure 27 shows the results of an intracellular cytokine production assay in CD4+, CD8, or DN T cells among lymphocytes expanded from pancreatic cancer lesions. The graph in FIG. 8A shows the percentage of T cells producing the cytokines IFN γ (upper panel) and TNF α (lower panel) following stimulation with a tumor associated antigen, either mesothelin, NY-ESO-1 or survivin, in CD4+ (left panel), CD8+ (middle panel) and the DN subpopulation (right panel). FIG. 8B shows an example of flow cytometry analysis of NY-ESO-1 stimulation. T cells were gated on Side Scatter (SSC) with CD3+ and then CD8+ versus IFN γ (upper panel) or TNF α (lower panel) production. This data indicates that IL-2, IL-15 and IL-21 amplified TILs from pancreatic cancer patients show strong reactivity to the common tumor antigen, NY-ESO-1.

Figure 28 shows the results of an intracellular cytokine production assay in CD4+, CD8, or DN T cells in lymphocytes expanded from glioblastoma disease following stimulation with autologous tumor cells. The graph in fig. 9A shows the percentage of T cells producing the cytokines IFN γ and TNF α, CD4+ T cells, left graph, after stimulation with autologous tumor cells; CD8+ T cells (middle panel) and DN T cells (right panel). Fig. 9B shows an example of flow cytometric analysis of cells stimulated with autologous tumor cells. Relative to IFN γ (upper panel) or TNF α (lower panel) production, T cells were gated with CD3+ and then with CD4+ (upper panel) or CD8+ (lower panel) in Side Scatter (SSC). This data indicates that IL-2, IL-15 and IL-21 expanded TIL from glioblastoma patients showed strong reactivity to autologous tumor cells.

FIG. 29 shows the results of an intracellular cytokine production assay measuring TNF α production from lymphocytes expanded with cytokine mixtures of IL-2, IL-15 and IL-21. The upper panel shows the positive control (maximal stimulation). The panel in (a) shows the results of cytokine production by CD4+ gated expanded T cells in response to autologous tumor cells (left: all TILs, right: TILs gated with VB2+ T cells). The following figures: background generation in the entire TIL population (left) and VB2+ TIL (right). This data indicates that the TCR VB family (here TCR VB2) that preferentially expands in IL-2, IL-15, and IL-21TIL is directed against autologous tumor cells.

Figure 30 shows levels of INF γ in lymphocytes expanded from pancreatic tumor tissue after stimulation. TIL + tumors represent lymphocytes stimulated to expand with autologous tumor cells. TIL + OKT3 represents the stimulation of lymphocytes with the CD3 antibody. W6/32 is an antibody that blocks CD8+ TIL. Antibody L243 blocks CD4+ TIL. This data indicates that IL-2, IL-15 and IL-21 amplified TIL are specific for autologous tumors in patients.

Fig. 31 shows the results of an analysis of cytolytic response of TILs expanded from glioblastoma patients to autologous tumor cells. The numbers on the X-axis represent the ratio of TIL to tumor cells. The percentage on the Y-axis represents the number of tumor cells killed after 4 hours of treatment with expanded TIL, measured by radioactive release.

Fig. 32 shows the results of an analysis of cytolytic response of expanded monoclonal T cells and/or preferentially expanded TILs from glioblastoma patients to autologous tumor cells. The numbers on the X-axis represent the ratio of TIL to tumor cells. The percentage on the Y-axis represents the number of tumor cells killed after 4 hours of treatment with expanded TIL, measured by radioactive release.

FIG. 33 shows the results of an analysis of cytolytic response of TILs expanded from pancreatic cancer patients to autologous tumor cells. The numbers on the X-axis represent the ratio of TIL to tumor cells. The percentage on the Y-axis represents the number of tumor cells killed after 4 hours of treatment with expanded TIL, measured by radioactive release. These IL-2, IL-15 and IL-21 expanded TILs display a very focused TCR lineage and show a very strong cytotoxic response to autologous tumor cells.

Detailed Description

The present inventors have found that the combination of interleukin IL-2, interleukin IL-15 and interleukin IL-21 provides a significant improvement in immunotherapy using lymphocytes. A major advantage is that the expansion and stimulation of patient-derived lymphocytes with a composition of a combination of at least two types of cytokines selected from the group consisting of IL-2, IL-15 and IL-21 is particularly beneficial for the production of clinically relevant lymphocytes, in particular T cells.

The "clinically relevant lymphocytes" of the invention are specific for and interact with clinically relevant antigens. There are three groups of clinically relevant lymphocytes, namely tumor-reactive lymphocytes, infectious disease-reactive lymphocytes and autoimmune disease-reactive lymphocytes.

"clinically relevant lymphocytes" are also known as antigen-editing lymphocytes. The term clinically relevant is also used for a subset of lymphocytes. Particularly preferred clinically relevant lymphocytes are clinically relevant T cells or antigen-edited T cells.

The "clinically relevant antigen" of the present invention is an antigen involved in a disease. Thus, the clinically relevant antigen may be a tumor associated antigen, TAA, a Pathogen Associated Antigen (PAA) or an autoantigen. Tumour-reactive lymphocytes are specific for and interact with TAA. Infectious disease reactive lymphocytes are specific for and interact with PAA, and autoimmune disease reactive lymphocytes are specific for and interact with self antigens.

An "antigen" (Ag) of the present invention is any structural substance that serves as a target for a receptor, TCR, or antibody, respectively, of an adaptive immune response. Antigens are in particular proteins, polysaccharides, lipids and their substructures such as peptides. Lipids and nucleic acids are particularly antigenic when combined with proteins or polysaccharides.

"pathogen-associated antigen" (PAA) refers to a portion of a pathogen, such as bacteria, viruses, and other microorganisms, such as capsules, cell walls, flagella, and toxins.

An "autoantigen" is typically a complex of peptides, oligopeptides, polypeptides or proteins from an individual that are recognized by the immune system of the same individual. This effect often leads to autoimmune diseases.

The "tumor associated antigen" or "TAA" of the present invention is an antigen presented by MHC I or MHC II molecules or atypical MHC molecules on the surface of tumor cells. As used herein, TAA includes "tumor-specific antigens" found only on the surface of tumor cells, but not on the surface of normal cells.

As shown in the examples, the use of a combination of IL-2, IL-15 and IL-21 is capable of specifically inducing the proliferation of clinically relevant lymphocytes in a body sample obtained from a patient. The methods of the invention provide an easy protocol for expanding clinically relevant lymphocytes. This solution is particularly advantageous compared to the solutions of the state of the art, since dendritic cells are not required. Furthermore, the present inventors could demonstrate that the lymphocyte population obtained after expansion with a cytokine mixture of a combination of at least two types of cytokines selected from the group consisting of IL-2, IL-15 and IL-21 comprises a lymphocyte composition that is advantageous for clinical use. For example, the composition has a high percentage of T_H1Helper T cells, and hardly contains T_H2Helper T cells. Another advantage is that regulatory T cells do not expand significantly, which may inhibit the therapeutic effect of the expanding lymphocyte population.

Thus, according to a first aspect, the present invention provides a composition for expanding lymphocytes, the composition comprising at least two types of cytokines selected from interleukin 2(IL-2), interleukin-15 (IL-15) and interleukin 21 (IL-21).

IL-2, IL-15 and IL-21 are members of the cytokine family, each having four alpha helical bundles. IL-2 has a key role in the key functions of the immune system, tolerance and immunity, primarily through its direct effects on T cells. IL-2 induces T cells to proliferate and differentiate into effector T cells and memory T cells.

IL-15 is a cytokine similar in structure to IL-2. Like IL-2, IL-15 binds to and signals through a complex of IL-2/IL-15 receptor beta chains. IL-15 induces T cell activation and proliferation of particularly CD8+ T cells (30), and also provides survival signals to maintain memory cells in the absence of antigen, favoring CD8+ T cells and activating monocytes. IL-15 appears to drive the proliferation of immune effector T cells and prevent tumor-associated immunosuppression (31).

IL-21 is a cytokine with a strong regulatory effect on immune system cells, including Natural Killer (NK) cells and cytotoxic T cells. IL-21 enriches central memory T cells with the phenotype CD28+ CD127hi CD45RO + and enhances the cytotoxicity of cytotoxic T cells. IL-21 can maintain T cells at an early stage in their differentiation and maturation (35).

According to the invention, the composition of the combination of at least two types of cytokines selected from the group consisting of IL-2, IL-15 and IL-21 is also referred to as "cytokine mixture".

As used herein, "interleukin 2" or "IL-2" refers to human IL-2 as defined by SEQ ID NO:1 and functional equivalents thereof. Functional equivalents of IL-2 include related substructures of IL-2 or fusion proteins that retain IL-2 function. Thus, IL-2 is defined to include a sequence identical to SEQ ID NO:1, preferably at least 90%, more preferably at least 95%, most preferably at least 98% sequence identity. Recombinant human IL-2 produced in E.coli as a single, non-glycosylated polypeptide chain of 134 amino acids and a molecular weight of 15kDa is commercially available in lyophilized form from Prospec as CYT-209.

As used herein, "interleukin 15" or "IL-15" refers to human IL-15 and functional equivalents thereof. Functional equivalents of IL-15 include related substructures or fusion proteins of IL-15 that retain IL-15 function. Thus, IL-15 is defined to include a sequence identical to SEQ ID NO: 2, preferably at least 90%, more preferably at least 95%, most preferably at least 98% sequence identity. Recombinant human IL-15 produced in E.coli as a single, non-glycated polypeptide chain of 114 amino acids (and an N-terminal methionine) and a molecular weight of 12.8kDa is commercially available from Prospec as CYT-230 in lyophilized form.

As used herein, "interleukin 21" or "IL-21" refers to human IL-21 and functional equivalents thereof. Functional equivalents of IL-21 include related substructures or fusion proteins of IL-21 that retain IL-21 function. Thus, IL-21 is defined to include a sequence identical to SEQ ID NO: 3, preferably at least 90%, more preferably at least 95%, most preferably at least 98% sequence identity. Recombinant human IL-21 produced in E.coli as a single, non-glycosylated polypeptide chain of 132 amino acids and a molecular weight of 15kDa is commercially available in lyophilized form from Prospec as CYT-408.

As used herein, a "peptide" may be composed of any number of any type of amino acid, preferably naturally occurring amino acids, and preferably linked by peptide bonds. In particular, the peptide comprises at least 3 amino acids, preferably at least 5, at least 7, at least 9, at least 12 or at least 15 amino acids. Furthermore, there is no upper limit on the length of the peptide. Preferably, however, the peptides of the invention are not more than 500 amino acids in length, more preferably not more than 300 amino acids in length; even more preferably not more than 250 amino acids.

Thus, the term "peptide" includes "oligopeptides", which generally refer to peptides of 2 to 10 amino acids in length, and "polypeptides", which generally refer to peptides of greater than 10 amino acids in length.

The term "protein" refers to a peptide having at least 60, at least 80, preferably at least 100 amino acids.

The term "fusion protein" of the present invention refers to a protein produced by joining two or more genes, cdnas or sequences that originally encode separate proteins/peptides. The genes may be naturally occurring in the same organism or in different organisms or may be synthetic polynucleotides.

The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". For The purposes of The present invention, The degree of sequence identity between two amino acid sequences is determined using The Needleman-Wunsch algorithm (Needleman and unscch, 1970, J.Mol.biol.48:443-453) as performed in The Needle program (preferably version 3.0.0 or more) of The EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et a/, 2000, Trends Genet.16: 276-277). The optional use parameters are: gap opening penalty of 10, gap extension penalty of 0.5, and EBLOSUM62 (BLOSUM 62 version EMBOSS). The output of Needle labeled "longest identity" (obtained using the thenobrief option) is used as the percent identity and is calculated as follows:

(same residue X100)/(alignment length-total number of gaps in alignment).

The transitional term "comprising" synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional unrecited elements or method steps. The transitional phrase "consisting of" excludes any elements, steps, or components not specified in the claims except for impurities normally associated therewith. The phrase "consisting of" when it appears in a clause of the subject matter of the claims, rather than immediately following the preamble, it only restricts the elements listed in the clause; other elements are not excluded from the claims as a whole. The transitional phrase "consisting essentially of" limits the scope of the claims to the specific materials or steps of the claimed invention "as well as those that do not materially affect the basic and novel characteristics. A claim consisting of ' is in an intermediate zone between a closed claim written in ' consisting of … … ' format and a fully open claim written in ' comprising ' format.

As used herein, "amplification" or "clonal amplification" refers to the generation of all daughter cells originally derived from a single cell. In clonal expansion of lymphocytes, all progeny share the same antigen specificity.

According to one embodiment of the invention, the composition according to the first aspect comprises two or three types of cytokines. Other cytokines may interfere with the amplification results provided by the compositions of the invention.

Alternatively, in addition to the combination of IL-2, IL-15 and IL-21, other cytokines used may positively affect the lymphocyte population. Thus, the composition of the first aspect of the invention may comprise further cytokines in addition to IL-2, IL-15 and IL-21. Examples are IL-1. beta. IL-4, GM-CSF, IL-12, IL-8, IL-17, TNF. alpha., IL-32. IL-1. beta. is involved in priming (priming), differentiation to effector B cells or T cells upon first exposure to a specific antigen. IL-4 and GM-CSF are involved in dendritic cell stimulation and/or sensitization. IL-12 involvement in T_H1And (4) reacting. IL-18 stimulates γ δ -T cells. IL-17 and TNF α play pro-inflammatory roles. IL-32 also plays a pro-inflammatory role in favor of a long-term protective immune response.

According to one embodiment of the first aspect, the composition comprises IL-2 and IL-15. The composition may also comprise IL-2 and IL-21. Alternatively, the composition may comprise IL-15 and IL-21. Although two of the cytokines IL-2, IL-15 and IL-21 may already be sufficient to obtain a clinically relevant lymphocyte population, it is preferred to use all three cytokines in the composition.

According to another embodiment, the composition of the first aspect is in liquid form. In particular, the composition is a cell culture medium. Any known cell culture medium is possible according to the invention. A non-limiting example of a cell culture medium is a synthetic medium, which is a medium derived from serum, plasma, or whole blood, or any combination thereof.

According to another embodiment, the concentration of IL-2 in the liquid composition is in the range of 10 to 6000U/ml. International units (U) are standard measures of the amount of IL-2. It is determined by the ability to induce proliferation of CTLL-2 cells. Concentrations below 10U/ml are too low to achieve any significant effect. Concentrations above 6000U/ml may have cytotoxic effects. The concentration of IL-2 is preferably in the range of 500 to 2000U/ml. More preferably, the concentration of IL-2 is in the range of 800 to 1100U/ml. As shown in the examples, the optimum results were obtained with a concentration of about 1000U/ml.

According to another embodiment of the first aspect, the concentration of IL-15 is in the range of 0.1 to 100 ng/ml. The concentration range follows the same rationale as IL-2. Concentrations below 0.1ng/ml are not considered to have any significant effect on the cells. Concentrations above 100ng/mL may have cytotoxic effects. Preferably, the concentration of IL-15 is in the range of 2 to 50ng/ml, more preferably in the range of 5 to 20 ng/ml. The most preferred concentration is about 10 ng/ml.

In another embodiment, the concentration of IL-21 is in the range of 0.1ng/ml to 100ng/ml, preferably in the range of 2 to 50ng/ml, more preferably in the range of 5 to 20 ng/ml.

It is understood that any of these concentration ranges for one of the cytokines may be combined with any of the concentration ranges for the other cytokines according to the present invention.

According to one embodiment, the combination comprises a mixture of IL-15 and IL-21. The mixture preferably comprises IL-15 and IL-21 in each case in the range from 10 to 100 ng/ml. IL-15 and IL-21 may provide synergistic effects, particularly on subpopulations of lymphocytes in precursor, memory and effector cell populations.

According to one embodiment of the first aspect, the combination comprises IL-2 at a concentration of 800 to 1000U/ml and IL-15 and IL-21 both at a concentration of 5 to 20 ng/ml. According to another embodiment, the combination comprises IL-2 at a concentration of about 1000U/ml and IL-15 and IL-21 both at a concentration of about 10 ng/ml.

The combination of IL-2, IL-15 and IL-21 is particularly useful for promoting expansion of clinically relevant lymphocytes in lymphocyte compositions, particularly in patient samples. As shown in the examples, the present inventors developed methods for preparing specific clinically relevant lymphocytes from patient samples.

The combination of IL-2, IL-15 and IL-21 is particularly useful for promoting expansion of clinically relevant lymphocytes in lymphocyte compositions, particularly in patient samples. As shown in the examples, the present inventors developed a method of reducing the frequency of PD1 and LAG3+ T cells, wherein PD1 and/or LAG3 expression is used as a marker of T cell depletion, rather than as a marker of antigen-experienced T cells.

The combination of IL-2, IL-15 and IL-21 is particularly useful for promoting expansion of clinically relevant lymphocytes in lymphocyte compositions, particularly in patient samples. As shown in the examples, the present inventors developed a method of increasing the frequency of 4-1BB expression, wherein 4-1BB expression was used as a marker for T cells that the antigen experienced.

Thus, according to a second aspect, the present invention provides a method of preparing a clinically relevant lymphocyte population, comprising the steps of:

-obtaining a body sample, in particular a tissue sample or a body fluid sample, comprising at least one lymphocyte from a mammal and optionally isolating cells in the body sample,

-culturing a body sample in vitro to expand and/or stimulate lymphocytes in the sample, wherein culturing comprises the use of at least two types of cytokines selected from the group consisting of IL-2, IL-15 and IL-21,

and optionally determining the presence of clinically relevant lymphocytes in the cultured sample.

At least two types of cytokines selected from the group consisting of IL-2, IL-15 and IL-21 are preferably used in the concentrations defined above. Preferably, all three cytokines IL-2, IL-15 and IL-21 are used together.

As shown in the examples, the methods can be used to generate a population of tumor-reactive lymphocytes, a population of autoimmune disease-reactive lymphocytes, or a population of infectious disease-reactive lymphocytes. Preferably, the lymphocyte population produced by the method of the second aspect is a tumour reactive lymphocyte population.

Lymphocytes will generally comprise a variety of different lymphocytes. In these, the lymphocytes may be lymphocytes present with suitable receptors for interacting with clinically relevant antigens, in particular tumor-associated antigens, infectious disease-associated antigens or autoimmune disease-associated antigens. Such clinically relevant lymphocytes are particularly strongly expanded using the method of the invention. However, other lymphocytes not specific for a clinically relevant antigen are also amplified in the method of the invention.

Thus, the result of culturing cells from a body sample with a composition comprising IL-2, IL-15 and/or IL-21 results in the formation of a lymphocyte population comprising a clinically relevant lymphocyte population. It is possible, but not a necessary step of the method of the second aspect of the invention, to determine the presence of clinically relevant lymphocytes in the cultured sample. This step helps to verify that the expanded lymphocyte population is indeed useful as a therapy (therpeutic).

The body sample may be taken from any part of the body containing lymphocytes. Examples of body samples are peripheral blood, cord blood, bone marrow, lymph nodes, liver, pleural effusion, thorax, abdominal cavity, synovial fluid, peritoneum, retroperitoneal space, thymus and tumors.

A sample of tumor-derived lymphocytes is also known as Tumor Infiltrating Lymphocytes (TILs). As used herein, "TIL" is an abbreviation for "tumor infiltrating lymphocytes". TILs are lymphocytes of any kind located in, on or around a tumor. TILs are lymphocytes of any kind located in and around tumors.

Because they are located in tumors, TILs may have experienced tumor-associated antigens. Thus, clinically relevant lymphocytes, particularly tumor reactive lymphocytes, can be expanded using the method of the invention without expansion of the antigen.

Samples from peripheral blood are also known as Peripheral Blood Mononuclear Cells (PBMCs). Depending on the type of disease, different body samples may be preferred.

The term "mammal" as used herein refers to any mammal, including but not limited to mammals of the order rodentia such as mice and hamsters and mammals of the order lagomorpha (order logomorph) such as rabbits. Preferably, the mammal is from the order carnivora, including felines (cats) and canines (dogs). More preferably, the mammal is from the order artiodactyla, including Bovine (cattle) and porcine (Swine); or order Perssodactyla (Perssodactyla), including equine (horse). Most preferably, the mammal is an animal of the order primates, quadrupeds (Ceboids) or simians (Simoids) (monkeys) or apes (humans and apes). A particularly preferred mammal is a human.

According to one embodiment of the second aspect, the mammal from which the body sample is obtained is a human. The mammal may be suffering from or at risk of suffering from a neoplastic disease. The risk of developing a neoplastic disease includes high risk, moderate risk and low risk. Mammals suffering from such precancerous conditions, for example, have precancerous lesions, which are morphologically atypical tissues, appear abnormal under microscopic examination, and are more likely to develop cancer in the precancerous lesion than their apparently normal counterparts.

In addition, the mammal may have or be at risk of having an infectious disease. The risk of contracting an infectious disease includes high risk, moderate risk and low risk. The mammal may also have or be at risk of developing an autoimmune disease. Risks of developing autoimmune diseases include high risk, moderate risk, and low risk. For example, when the host has certain genetic mutations (IFN γ receptor deficiency, or acquired antibodies against cytokines such as IL-12 or IFN γ), the risk of getting intracellular infections (CMV, EBV, TB, HPV) is high. An intermediate risk is immunosuppression when treating patients with corticosteroids or with anti-TNF α or TNF α receptor-directed agents. The low risk may be a co-infection with other pathogens or a temporary reduction in immunity during major surgery. Similar examples are multiple sclerosis, rare neurological diseases such as narcolepsy, rheumatoid arthritis and chronic autoimmune diseases associated with the gastrointestinal system-different clinical manifestations associated with genetic markers.

If the mammal is suffering from or at high risk of suffering from a tumor disease, the preferred body sample is peripheral blood or the tumor itself. As shown in the examples, lymphocytes from peripheral and tumor sources can be treated with the methods of the invention to obtain strong anti-tumor properties. If the disease is an autoimmune disease, the preferred body sample is peripheral blood. Furthermore, when the disease is an infectious disease, the preferred body sample is also peripheral blood. As shown in the examples, clinically relevant lymphocytes can be expanded from peripheral blood in these cases. Without being bound by theory, it is assumed that the peripheral blood contains lymphocytes that have been exposed to a clinically relevant antigen, such as a clinically relevant antigen on a tumor or infection.

Culturing a body sample in vitro to expand and/or stimulate lymphocytes may comprise one or more substeps. Thus, in one embodiment, the in vitro culture comprises a first expansion step comprising incubation in a medium comprising IL-2, IL-15 and IL-21 until lymphocytes become detectable.

By "detectable" in the context of the present invention is meant that the lymphocytes become visible, for example, particularly by microscopy. To reach 5X 10³At individual cell/ml concentrations, lymphocytes are typically detected using a standard light microscope.

The detection of lymphocytes may comprise any method known in the art that is capable of detecting the presence of lymphocytes above a certain threshold. The purpose of the first amplification step is to gently induce cell proliferation and stimulate cells with a cytokine mixture.

The incubation time for the first amplification step is in the range of 6 hours to 180 days. The large range of incubation times is due first to the fact that samples from different donors may appear very different. It has also been shown that lymphocytes from different body samples have widely different growth rates. For example, tumors directly derived from glioblastoma or pancreatic cancer have widely different lymphocyte growth. Lymphocytes from pancreatic cancer are already detectable within 2 to 5 days. Lymphocytes derived from glioblastoma are detectable after one to two weeks. Thus, lymphocytes from other body samples may take even longer to become detectable.

Preferably, the incubation time of the first amplification step is in the range of 4 days to 10 days. For peripheral blood cells, an incubation time of about 7 days has proven to be particularly beneficial for other expanded results. However, as described above, depending on the sample, only 4 days of amplification may be sufficient, or on the other hand, about 10 or more days may be required. The incubation time is preferably 6-8 days, in particular about 7 days, due to the good results of the PBMCs shown in the examples.

According to one embodiment of the invention, the medium of the first amplification step comprises at least one amplification antigen. The amplified antigen is a known clinically relevant antigen or a fragment thereof, mutant thereof (mutant) or variant thereof (variant). As used herein, "mutant" is defined as an amino acid sequence that differs from a reference sequence by the insertion, deletion, or substitution of at least one amino acid. The amplification antigen is preferably selected from the group consisting of TAA, PAA and autoantigens.

Preferably, the method of the invention comprises amplifying multiple copies of the antigen (multiple copies). The increased copy number of the amplified antigen results in an increased rate of expansion of clinically relevant lymphocytes, particularly T cells.

In one embodiment of the invention, the medium of the first amplification step comprises a plurality of amplification antigens. Preferably, the plurality of amplified antigens comprises known clinically relevant antigens and one or more mutants of clinically relevant antigens. In addition to the antigen itself, MHC class I/peptides, in particular peptides, which present the antigen may also be mutated. Using one or more wild-type, variant or mutant of a clinically relevant antigen or MHC class I molecule as an amplification antigen results in a diverse panel of lymphocytes, in particular T cells reactive against the (nominal) clinically relevant antigen.

According to a preferred embodiment of the method according to the second aspect, the body sample is a tumor and no amplification antigen is used in the culture.

Prior to isolating the cells in the tumor sample, the tumor tissue is preferably washed twice.

In another preferred embodiment of the invention, the medium of the first amplification step comprises a plurality of amplification antigens. By having multiple antigens, the T cell products react with a more diverse T cell receptor lineage (receptor), as demonstrated by V β usage (V β -usage) associated with the use of a stimulating antigen.

In another preferred embodiment of the invention, the medium of the first amplification step comprises a plurality of amplification antigens. Stimulation of T cells from peripheral blood results in T cells recognizing multiple epitopes defined by cytotoxicity. When natural T cells are selected from the blood and stimulated with such antigens, they result in recognition of multiple epitopes of the antigen.

This is in contrast to pre-stimulated T cells or T cells from tumors, which tend to recognize a more limited or standard number of epitopes.

The choice of the amplified antigen depends on the disease to be treated. In particular, if the expanded population of clinically relevant lymphocytes is to be used against a tumor disease, the amplified antigen added to the first amplification step is preferably TAA. Alternatively, if the disease to be treated is an infectious disease, the amplified antigen added in the first amplification step is PAA. Furthermore, if the clinically relevant lymphocyte population is to be used for the treatment of an autoimmune disease, the expanded antigen is preferably an autoantigen.

It is believed that the expanded antigen in the first expansion step results in already stimulated clinically relevant lymphocytes in the cell mixture at an early stage, and thus it enhances the expansion of clinically relevant lymphocytes together with the cytokine mixture. This stimulation is also called antigen activation or antigen editing.

The first expansion step may be followed by a second expansion step wherein the cells are incubated with feeder cells and/or antibodies against CD3 in addition to at least two types of cytokines selected from the group consisting of IL-2, IL-15 and IL-21. Expansion with feeder cells and antibodies against CD3 has been described in the prior art. It is believed that feeder cells lead to improved cell growth. Feeder cells are irradiated cells that do not proliferate or proliferate only to a small extent (irradiated cells). Feeder cells increase the number of cell contacts in the culture and additionally feed (feed) the proliferating and expanding cell culture. The feeder cells are preferably in irradiated PBMCs. Allogeneic feeder cells are derived from an organism that is different from the mammal to be treated with the expanded clinically relevant lymphocytes. The autologous feeder cells are from the mammal to be treated.

The antibody against CD3 is preferably an antibody defined as OKT 3. OKT3 is a murine monoclonal antibody of the immunoglobulin IgG2a isotype. The target of OKT3 is CD3, and CD3 is a multimolecular complex found only on mature T cells. The interaction between T cells, OKT3 and monocytes results in T cell activation in vitro.

Preferably, the feeder cells are used in combination with CD3 and the cytokines IL-2, IL-15 and IL-21. According to one embodiment of the method of the second aspect, the ratio of feeder cells to lymphocytes is in the range of 1:1 to 1: 100. Preferably, the ratio of feeder cells to lymphocytes is in the range of 1:2 to 1: 50. As shown in the examples, a very low proportion of feeder cells is sufficient for the robust expansion of clinically relevant lymphocytes, in particular clinically relevant T cells.

In the examples, the ratio of 1:10 is sufficient to support the growth and expansion of lymphocytes. Values in the range of 1:5 to 1:20 are therefore not believed to lead to different results. The low number of feeder cells has at least two advantages. First, there is less interfering cell signal, allowing for more uniform and reliable amplification results. Secondly, fewer feeder cells result in fewer foreign substances in the immunotherapeutic product, i.e. the clinically relevant lymphocyte population, obtained by this method.

The second amplification step optionally further comprises amplifying the antigen in a culture medium. Preferably, no clinically relevant antigen or fragment is added to the medium of the second amplification step.

According to a preferred embodiment of the second aspect of the invention, the method comprises a refocusing step. The refocusing step comprises culturing in a medium comprising the refocused cells. Refocusing cells are cells from a mammal, particularly a human, from which a body sample is taken. Thus, the refocused cells are autologous cells that have been treated with at least one refocusing antigen. Any of the amplification antigens defined herein may also be used as a refocusing antigen. Preferably, in the method, the one or more refocusing antigens are the same as the one or more amplification antigens.

A refocused cell is a cell that has been incubated with the refocused antigen for at least 30 minutes or longer, e.g., at least 1 hour, at least 2 hours, at least 5 hours, or at least 10 hours. After incubation with the refocusing antigen, the refocusing cells are irradiated with at least 40 Gy. Preferably, the cells are irradiated with an intensity of at least 45Gy or more, for example at least 50Gy, particularly preferably 55 Gy. Due to this treatment, antigen-specific T cells are more efficiently expanded, recognize tumors, pathogens, or autoimmune cells, and the treatment can confer protection against cancer cells or premalignant lesions.

The time for the re-concentration step is in the range of 1 to 6 days, preferably 1 to 3 days. Although the refocusing step can be quite short, it significantly improves the yield of expanded clinically relevant lymphocytes, especially for clinically relevant lymphocytes expanded from peripheral blood.

Furthermore, the number of cells in the refocus is considerably low compared to the number of lymphocytes. In particular, the ratio of cells to lymphocytes in the refocusing is in the range of 1:1 to 1: 100. It was found that the best results are achieved if a first amplification step is followed by a re-concentration step and then by a second amplification step. The sequence of culture steps is particularly useful for generating populations of clinically relevant lymphocytes, particularly T cells.

According to one embodiment of the method, the first amplification step comprises the simultaneous addition of IL-2, IL-15 and IL-21 to the cell culture. To this end, a mixture of IL-2, IL-15 and IL-21 may be prepared and added to the cell culture medium, or IL-2, IL-15 and IL-21 may be added separately but simultaneously to the cell culture medium. As shown in the examples, the simultaneous application of IL-2, IL-15 and IL-21 results in an expanded lymphocyte population with a preferred lymphocyte composition.

To alter the composition of the expanded lymphocyte population, in the method of the invention, IL-21 may first be added to the cell culture medium alone in the first expansion step. After the addition of IL-21, IL-15 and IL-2 may be added simultaneously or sequentially. Preferably, IL-15 is added a second time and IL-2 is added last. In alternative embodiments, IL-15 is added as the first cytokine, followed by simultaneous addition of IL-2 and IL-21 or sequential addition of IL-2 and IL-21. For example, IL-21 is added a second time and IL-2 is added last.

The medium of the first amplification step and/or the second amplification step may comprise at least one amplification antigen. For example, the amplified antigen may be a fragment of a known TAA. Possible TAAs as amplification antigens are, for example, NY-ESO-1, tyrosinase tumor antigen, tyrosinase-related protein (TRP) -1, TRP-2, VEGFR-2 and members of the MAGE protein family, telomerase, p53, HER2/neu, mesothelin, carcinoembryonic antigen, survivin, EGFRvIII, VEGF, CAMPATH 1-antigen, CD22, CA-125, mucin-1, alpha-fetoprotein (alpha-1-getoprotein), PSMA.

In particular, fragments of TAA are peptides. Such a peptide may be, for example, a peptide comprising a sequence identical to SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO:7, and a peptide of at least 8 contiguous amino acids of an amino acid sequence that is at least 80% identical in amino acid sequence.

SEQ ID NO:4 is the amino acid sequence of known tumor associated antigen NY-ESO-1. SEQ ID NO:5 is the amino acid sequence of the known tumor associated antigen survivin. SEQ ID NO:6 is the amino acid sequence of the known tumor associated antigen mesothelin. SEQ ID NO:7 is the amino acid sequence of the tumor associated antigen EGFRvIII.

The amplification antigen may be, for example, a fragment of known PAA. Possible PAAs amplification antigens are, for example, CMVpp65 or EBV (EBNA-3, EBNA-1), HPV-16/33E6, E7 or L1. Fragments of PAA are in particular peptides. Such a peptide may for example be a peptide comprising a sequence identical to SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO:10 or SEQ ID NO:11, a peptide of at least 8 consecutive amino acids of an amino acid sequence which is at least 80% identical in amino acid sequence to the amino acid sequence of SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO:10 and SEQ ID NO:11 are the amino acid sequences of the known pathogen-associated antigens CMVpp65, EBNA-3, EBNA-1 and HPV-L1, respectively.

The amplification antigen may be, for example, a fragment of known PAA. Possible autoantigens as amplification antigens are, for example, PRDM2, UCHL3, INO80E, SLC12a6 and silk fibroin (Reelin). Fragments of PAA are in particular peptides. Such a peptide may for example be a peptide comprising a sequence identical to SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO:14 or SEQ ID NO: 15, a peptide of at least 8 consecutive amino acids of an amino acid sequence which is at least 80% identical in amino acid sequence to the amino acid sequence of SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO:14 and SEQ ID NO: 15 are the amino acid sequences of the autoantigens PRDM2, INO80E, UCHL3 and dnase b, respectively.

Additional components such as other cytokines may be added during the culturing step, particularly during the first or second amplification step or the re-concentration step.

Furthermore, promoter compounds may be added during the culturing step, in particular during the first or second amplification step or during the refocusing step. Preferably, an accelerator compound is added in the first amplification step. Promoter compounds as used herein are compounds that cause an increase in a specific lymphocyte subpopulation during expansion. A preferred accelerator compound is zoledronic acid. Zoledronic acid promotes the expansion of γ δ T cells. Another preferred enhancer compound enhances the expansion of B cells.

According to one embodiment, the co-stimulatory compound is added during the culturing step, in particular during the first or second amplification step or during the refocusing step. Co-stimulatory compounds are, for example, CD28 ligands that mediate T cell signaling. Examples of CD28 ligands mediating T cell signaling are members of the B7 superfamily (superfamilies), in particular B7-1(CD80) and B7-2(CD 86).

According to one embodiment of the method of the second aspect, detecting the presence of clinically relevant lymphocytes in the expanded lymphocyte sample comprises the use of an evaluation antigen (evaluation antigen).

For detection, the lymphocyte population is incubated with an evaluation antigen. The evaluation antigen may be an antigen under the definition of an amplification antigen. For example, a known clinically relevant antigen or a fragment thereof is added as an evaluation antigen to a culture medium of a lymphocyte population to stimulate lymphocytes. After the incubation time is over, a parameter indicative of activation of clinically relevant lymphocytes is measured.

Preferably, the evaluation antigen is added to the lymphocytes in a form bound to the MHC I complex. For example, the evaluation antigen can be presented to lymphocytes that bind to MHC dextramer. MHC dextramer is a fluorescently labeled MHC multimer bound to the dextrose backbone.

The use of multimeric MHC structures has the advantage that multiple copies of an antigen can be presented to a single lymphocyte, thereby enhancing the simulation by evaluating the antigen. Alternatively, a known clinically relevant antigen can be presented to the cultured sample as an evaluation antigen in the form of cells expressing the clinically relevant antigen as a transgene, and in addition, at least partially genetically matched allogeneic cells presenting the clinically relevant antigen on the cell surface can be added to the expanded lymphocytes.

The parameter indicative of the presence of clinically relevant lymphocytes may for example be the amount of production of one or more cytokines, in particular the amount of IFN-. gamma.or TNF. alpha.production. Other parameters indicative of the presence of clinically relevant lymphocytes are increased cell proliferation, increased cytotoxicity, increased cell signaling, and/or intracellular phosphorylation. The determination of these parameters is known in the art and is illustrated in the examples.

Preferably, in addition to using evaluation antigens derived from known clinically relevant antigens, clinically relevant lymphocytes specific to the mammal to be treated can be detected. For this purpose, cells, in particular tumor cells, which originate from the same mammal as the expanded lymphocytes, are used for presenting the evaluation antigen. Using these autologous cells as an evaluation presentation antigen, the presence of clinically relevant lymphocytes specific for clinically relevant antigens, which are not necessarily known clinically relevant antigens, can be verified.

In one embodiment according to the second aspect of the invention, the evaluation antigen presented to the cultured sample is selected from the following forms: cells (autologous cells) derived from the same mammal as the cultured sample, in particular tumor cells, at least partially gene-matched allogeneic cells, in particular tumor cells, or cells expressing the clinically relevant antigen as a transgene.

According to another embodiment, a method of detecting the presence of clinically relevant lymphocytes comprises contacting the lymphocytes with at least one evaluation antigen and determining a change in one of: cytokine production, in particular IFN-. gamma.or TNF. alpha.production, cell proliferation, cytotoxicity, signaling and/or intracellular phosphorylation.

Testing of these parameters can be combined with flow cytometry and cell sorting. Thus, it is also possible to separate the clinically relevant lymphocyte population, in particular the tumour-reactive lymphocyte population, from the expanded lymphocyte population. The isolated population of clinically relevant lymphocytes can be further cultured or used directly for immunotherapy.

The method of the second aspect of the invention results in the formation of a population of lymphocytes, including a clinically relevant population of lymphocytes. The clinically relevant lymphocytes included in the expanded lymphocyte population can be any type of lymphocyte.

Lymphocytes include B cells, NK cells and T cells. According to one embodiment, the clinically relevant lymphocyte is a B cell. According to one embodiment, the clinically relevant lymphocyte is an NK cell.

According to a third aspect, the present invention provides a clinically relevant lymphocyte obtainable by the method of the second aspect, wherein the clinically relevant lymphocyte is a T cell, an NK cell or a B cell.

The T cells are preferably selected from helper T cells (T cells)_HCells or CD4⁺T cells), in particular T_H1Cell, cytotoxic T cell (T)_CCells or CD8⁺T cells) in particular CD8⁺CXCR3⁺T cells, memory T cells, in particular central memory T cells (T)_CMCells), stem cell-like memory T cells (T)_SCMCells) or peripheral memory cells (T)_PMCells), gamma-delta T cells (gamma delta T cells), NK-T cells, mucosa-associated constant T cells (MAIT), double negative T cells (CD 3)⁺CD4^-CD8^-T cells).

According to one embodiment, the clinically relevant lymphocyte is a lymphocyte expressing a molecule (e.g., CXCR3) that facilitates entry into a tissue, particularly a tumor or an infected or inflamed tissue. According to another embodiment, the clinically relevant lymphocyte is a lymphocyte enriched for any one of: markers of long-term memory (especially CD117 and c-kit) and of cytolytic immune cell responses (especially CD107 a). As explained, the expanded lymphocyte population comprises not only clinically relevant lymphocytes, but also other lymphocytes that do not recognize clinically relevant antigens.

It is believed that clinically relevant lymphocytes and other lymphocytes in the culture obtained by the method of the second aspect are involved in the therapeutic effect. According to one embodiment of the third aspect, the invention relates to lymphocytes obtained by the method of the second aspect, which express molecules and cytokines that promote the formation of lymphocyte combinations for medical applications, including immunotherapy. The combination of lymphocytes for medical applications preferably comprises T cell precursors, T_CM、T_SCMAnd/or T_PMA cell.

According to one embodiment, the lymphocytes obtained by the method of the second aspect express molecules and cytokines that promote the expansion of clinically relevant lymphocytes. According to another embodiment, the lymphocytes obtained by the method of the second aspect produce a cytokine selected from the group consisting of IFN- γ, TNF- α, IL-2, IL-17, and any combination thereof. According to one embodiment, the lymphocyte is a CD3+ CD4-CD8-T cell.

According to a fourth aspect, the present invention provides a population of lymphocytes obtained by the second aspect of the invention, comprising a population of clinically relevant lymphocytes.

The lymphocyte population may consist of a clinically relevant lymphocyte population. The clinically relevant lymphocyte population may be monoclonal, oligoclonal, or polyclonal.

In one embodiment, the clinically relevant lymphocyte population is polyclonal and responds to multiple antigens or to different epitopes of the same antigen. Preferably, the clinically relevant lymphocyte populations respond to different antigens.

Lymphocytes, particularly T cells that respond to multiple antigens, can prevent the risk of recurrence of pre-cancerous cells, tumor cells, and pathogens, thereby reducing the risk of immune escape.

The composition of the lymphocyte population of the fourth aspect is a distinct lymphocyte phenotype, particularly a T cell phenotype useful for immunotherapy.

The lymphocyte population has a low percentage of regulatory T cells. Regulatory T cells are known to inhibit the therapeutic function of lymphocyte populations. According to an embodiment of the fourth aspect, in the lymphocyte population, T is based on the total number of T cells_regIs less than 5%, preferably less than 3%.

In addition, most of the T's in the lymphocyte population_HThe cell comprises T_H1Phenotype. According to one embodiment of the invention, based on T_HTotal number of cells, T_H1The percentage of cells is at least 10%, preferably at least 50%, more preferably at least 70%.

As indicated by the increased percentage of CXCR3+ cells, the percentage of cytotoxic CD8+ T cells was increased in the lymphocyte population. According to one embodiment of the invention, the percentage of CXCR3+ cells is at least 10%, at least 50%, more preferably at least 70% based on the total number of CD8+ T cells.

In addition, the lymphocyte population may comprise an increased number of T cells recently contacted with its antigen, as identified, for example, by the 4-1BB + phenotype. According to one embodiment of the fourth aspect, the percentage of 4-1BB + T cells is at least 1%, preferably at least 2%, more preferably at least 2.5%, based on the total number of T cells. According to one embodiment, the percentage of CD107+ cells is at least 1%, preferably at least 2%, more preferably at least 2.5%, based on the total number of T cells. CD117+ T cells are associated with long-term memory populations.

CD8+ T cells of the CXCR3+ phenotype are also associated with invasiveness to tissues and thus may be particularly advantageous for immunotherapy against cancer. In addition, the lymphocyte population may comprise a sufficient percentage of CD3+ CD4-CD 8-cells. These double negative T cells are highly specific for antigen targets, as they rely on co-receptors CD4+ or CD8+ and produce inflammatory cytokines. Therefore, they are cytotoxic.

According to one embodiment, the lymphocyte population comprises a percentage of at least 1%, preferably at least 3%, more preferably at least 5% of CD3+ CD4-CD 8-cells, based on the total number of T cells. According to another embodiment, the percentage of γ δ T cells is at least 1%, preferably at least 3%, more preferably at least 5%, based on the total number of T cells in the lymphocyte population. γ δ T cells have the role of recognizing stressed cells, transformed cells, infected cells such as CMV + target cells. γ δ T cells also cross-recognize virally transformed cells.

According to one embodiment, the population of lymphocytes comprises the following characteristics:

-based on the total number of T cells, T_regIs less than 5%, preferably less than 3%;

based on T_HTotal number of cells, T_H1The percentage of cells is at least 50%, preferably at least 70%, more preferably at least 80%;

based on CD8⁺Total number of T cells, the percentage of CXCR3+ T cells is at least 50%, preferably at least 70%, more preferably at least 80%;

-the percentage of 4-1BB + T cells is at least 1%, preferably at least 2%, more preferably at least 2.5%, based on the total number of T cells;

-the percentage of CD117+ T cells is at least 1%, preferably at least 2%, more preferably at least 2.5%, based on the total number of T cells;

-the percentage of CD3+ CD4-CD 8-cells is at least 1%, preferably at least 3%, more preferably at least 5%, based on the total number of T cells; and

-the percentage of γ δ T cells is at least 1%, preferably at least 3%, more preferably at least 5%, based on the total number of T cells.

According to one embodiment, the percentage of precursor T cells (CD45RA + CCR7+) in the lymphocyte population is at least 1%, preferably at least 2%, more preferably at least 3%, based on the total number of T cells. Central memory T cells have been shown to be associated with the success of T cell therapy.

Central memory cells produce several cytokines that are therapeutically beneficial, providing a good memory response. However, central memory cells do not penetrate well into tissues. In one embodiment, the percentage of peripheral memory cells (CD45RA-CCR7-) is at least 2%, preferably at least 5%, more preferably at least 10%, based on the total number of T cells. Peripheral memory cells exhibit high cytokine production and penetrate well into tissues. Thus, these cells are particularly useful for immunotherapy against cancer.

Terminally differentiated T cells (CD45RA + CCR7-) are also able to enter tissues. In addition, these cells show production of cytokines that are therapeutically useful. According to one embodiment, the percentage of effector T cells (CD45RA + CCR7-) is at least 1%, preferably at least 3%, more preferably at least 5%, based on the total number of T cells. The percentage of precursor T cells (CD45RA + CCR7+) may be at least 1% based on the total number of T cells. Preferably, this percentage is at least 2%, more preferably at least 3%. Precursor T cells only show cytokine production and hardly enter tissues. However, these cells may be reversed T cells with lifelong memory effects.

The population of lymphocytes of the fifth aspect may also be characterized by: the amount of cytokine production in the cells after stimulation with the evaluation antigen is at least two times the standard deviation without evaluation antigen stimulation. Furthermore, stimulation with the evaluation antigen resulted in a value of CD107a induction that was at least 2-fold the standard deviation without evaluation antigen stimulation.

Because of these parameters, lymphocyte populations and/or clinically relevant lymphocyte populations represent immunotherapeutic products useful for the treatment of cancer, infectious diseases, and autoimmune diseases.

According to a fifth aspect, the present invention provides an immunotherapy for treating or preventing a neoplastic disease, an infectious disease or an autoimmune disease in a mammal, said immunotherapy comprising the steps of: generating a population of clinically relevant lymphocytes according to the second aspect of the invention, wherein the body sample is obtained from said mammal; and administering a clinically relevant population of lymphocytes to the mammal. The infectious disease may be any known infectious disease associated with any known pathogen.

The neoplastic disease of the invention may be any cancer, including any of the following: acute lymphocytic cancer, acute myelogenous leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer or pleural cancer, nasal cavity cancer or middle ear cancer, vulva cancer, chronic lymphocytic leukemia, chronic myelogenous cancer, cervical cancer, glioma, hodgkin's lymphoma, hypopharynx cancer, kidney cancer (kidney cancer), laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-hodgkin's lymphoma, ovarian cancer, peritoneal cancer, omentum cancer, mesenteric cancer, pancreatic cancer, pharynx cancer, prostate cancer, rectal cancer, kidney cancer (renal cancer), skin cancer, soft tissue cancer, testicular cancer, thyroid cancer, bladder cancer, and ureteral cancer such as esophageal cancer, gastric cancer (gasteric cancer), Pancreatic cancer, gastric cancer (stomach cancer), small intestine cancer, gastrointestinal carcinoid tumor, oral cancer, colon cancer and liver and gall bladder cancer. Preferably, the cancer is glioblastoma and pancreatic cancer.

To avoid immune effects against clinically relevant lymphocytes, cancer patients can be "conditioned" prior to administration of T cells. The regulation has three purposes. The first is to provide more space for the infused lymphocyte population, the second is to remove adverse effectors, and the third is to increase the production of growth factors that can facilitate rapid expansion and survival of T cells, i.e., autologous IL-7 and IL-15 production.

Clinically relevant lymphocytes can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure. In one embodiment, the clinically relevant lymphocytes can be autologous to the subject, i.e., the clinically relevant lymphocytes are obtained from the subject in need of treatment and then administered to the same subject.

Administration of autologous cells to a subject may result in reduced rejection of host cells as compared to administration of non-autologous cells. Alternatively, the host cell is an allogeneic cell, i.e., the cell is obtained from a first subject and administered to a second subject different from the first subject but belonging to the same species. For example, allogeneic clinically relevant lymphocytes may be derived from a human donor and administered to a human recipient that is different from the donor.

To practice the methods disclosed herein, an effective amount of a clinically relevant lymphocyte described herein, or a composition thereof, can be administered to a subject in need of treatment (e.g., a human cancer patient, a patient with an infectious disease, a patient with an autoimmune disease) by a suitable route, e.g., intravenous administration. The cells may be introduced by injection, catheter, or the like. Additional drugs (e.g., cytokines) may also be introduced together or sequentially, if desired. Any cell or composition thereof can be administered to a subject in an effective amount. As used herein, an effective amount refers to the amount of each agent (e.g., cell or composition thereof) that imparts a desired therapeutic effect to a subject upon administration. It will be apparent to one skilled in the art that the amount of the cells or compositions described herein can be determined to achieve the desired therapeutic effect. See, for example, references 2 to 5. As recognized by one skilled in the art, effective amounts vary according to: the particular condition being treated, the severity of the condition, individual patient parameters (including age, physical condition, size, sex, and weight), the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration, and similar factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount reduces, alleviates, ameliorates, reduces symptoms of, or delays progression of the expected disease or disorder in the subject.

By "administering" is meant physically introducing a cell-containing composition or pharmaceutical composition described herein into a subject using any of a variety of methods and delivery systems known to those of skill in the art.

Administration of the tumor-reactive lymphocyte population is preferably locally introduced near or into the patient's tumor. Alternatively, the population of tumor-reactive lymphocytes is administered into the blood circulation. Preferably, other clinically relevant lymphocytes are added to the blood circulation.

According to one embodiment of the invention, the dose of clinically relevant lymphocytes administered to the subject is a dose comprising at least about 4 x 10 per kilogram body weight⁶、4.5×10⁶、5×10⁶、5.5×10⁶、6×10⁶、6.5×10⁶、7×10⁶、7.5×10⁶、8×10⁶、8.5×10⁶、9×10⁶、9.5×10⁶、10×10⁶、12.5×10⁶、15×10⁶、20×10⁶、25×10⁶、30×10⁶、35×10⁶、40×10⁶、45×10⁶、50×10⁶、60×10⁶、70×10⁶、80×10⁶、90×10⁶And (4) cells.

Immunotherapy according to the fifth aspect, a population of clinically relevant lymphocytes

-causing regression of cancer cells in the mammal;

-interfering with the progression of a pre-cancerous lesion to a malignant lesion;

-causing rapid senescence of tumor cells or precancerous cells;

-causing removal of autoantigen positive cells;

-killing, causing growth arrest or containment of pathogens;

-interfering with cancer stem cells; and/or

-inducing growth arrest of cancer cells or cells expressing autoantigens.

According to a sixth aspect, the present invention provides a composition according to the first aspect of the invention for use in medical therapy, in particular for use in the treatment and prevention of infectious diseases, autoimmune diseases or neoplastic diseases.

According to one embodiment of the sixth aspect, the use comprises generating a population of clinically relevant lymphocytes using the method of the second aspect of the invention.

In one embodiment of the composition according to the sixth aspect, the use comprises an immunotherapy according to the fifth aspect of the invention. As mentioned above, the specific cytokines of the cytokine mixture do not necessarily have to be used simultaneously, but can be added at different time points.

According to a seventh aspect, the present invention provides a kit for use in medical treatment, in particular for use in the treatment or prevention of an infectious disease, an autoimmune disease or a neoplastic disease, wherein the kit comprises IL-2, IL-15 and IL-21. The kit also optionally comprises at least one of: components that stimulate TCRs are, inter alia, OKT3, costimulatory molecules, feeder cells, and peptides comprising the amino acid sequence of a clinically relevant antigen. Preferably, the kit comprises all the components mentioned above.

The invention is further defined by the following examples.

Examples

Example 1 lymphocyte expansion protocol Using peripheral blood mononuclear cells

A) Materials and instruments

i) Instrument for measuring the position of a moving object

24 well plate (Becton, Dickinson (BD), REF 353504)

Sterile scalpel

Sterile tweezers

Mediachine (tissue refiner), (BD, catalog number: 340588)

Medicon,50 μm, (BD, catalog number 340591)

Filcons,200 μm (BD, Cat. No.: 340613)

Laminar flow purification hood (2 level biological safety hood)

Low temperature freezer, -80 deg.C

Refrigerator with a door

Centrifugal machine

ii) articles

Gloves (emulsion)

Experiment work clothes

Sterile centrifuge tube, 15 mL.

Pipette tip (Pipet tips)

Assistant pipettor (Pipette aid)

Waste container

iii) reagents

RPMI 1640，(Gibco,REF 61870-044)

Cellgro，(CellGenix,Cat:20801-0100)

Mixed human AB serum (Innovative Research, IPLA-SerAB-13458)

Fetal bovine serum (Gibco, REF:26140-079)

CD3 antibody (OKT3), (Biolegend, Cat:317304)

Human IL-2(Prospec, catalog number: CYT-209-b)

Human IL-15(Prospec, Cat: cyt-230-b).

Human IL-21(Prospec, catalog number: cyt-408-b)

PEST (antibiotic)

Amphotericin

B) Process for producing a metal oxide

Apheresis (aphaeresis) was performed on healthy donors sensitized with NY-ESO-1 (primed). After separation of the blood components, the leukocyte-containing product is separated from the remaining components. Cells were suspended in Cellgro containing 5% pooled human AB serum, 1000U/ml IL-2, 10ng/ml IL-15 and 10ng/ml IL-21. The medium additionally contained 10. mu. mol of NY-ESO-1 peptide as an amplification antigen. Expansion of lymphocytes from peripheral blood comprises the step of stimulation with irradiated autologous PBMCs loaded with the antigen of interest. For this, autologous PBMC were loaded with 10mM NY-ESO-1 peptide for 2 hours at room temperature and then irradiated at 55 Gy. Loaded irradiated autologous PBMC were added to the lymphocyte cultures on day 7 and resuspended in fresh medium supplemented with 5% human pooled AB serum, with cytokine concentrations as defined above. On day 10, cells were expanded with Cellgro supplemented with 5% pooled human AB serum and cytokines. OKT3 antibody was added at a concentration of 30ng/ml in the same medium as above, and irradiated (55Gy) feeder cells, as well as peptides and cytokines, were added at 1:10 (feeder cells: T cells). On days 17 to 20 of culture, cells were harvested for analysis or transferred to patients.

Example 2 lymphocyte expansion protocol Using peripheral blood mononuclear cells

The procedure of example 2 is the same as that of example 1 except that the donor is a patient receiving NY-ESO-1 vaccination.

Example 3 lymphocyte expansion protocol Using peripheral blood mononuclear cells

Example 3 same as example 1, except that the donor is a patient whose tumor has expressed TAA NY-ESO-1.

Example 4 enrichment of Gamma Delta T cells in lymphocytes expanded with cytokines IL-2, IL-15 and IL-21

Amplification was performed according to the protocol described in example 1, with the difference that zoledronic acid was added to the cell culture at a concentration of 5 μ Mol. Zoledronic acid is known to promote expansion of TCR γ δ T cells. As shown in fig. 1, this experimental setup resulted in a strong increase in the frequency and absolute number of TCR γ δ T cells. Flow cytometry images of the expanded culture at different times are shown in figure 1. Thus, on the first day of expansion, 3.68% of the cells carried TCR γ δ. Already slightly increased the next day. Strikingly, almost 50% of T cells were the γ δ subpopulation after 7 days of expansion.

Example 5 expansion of lymphocytes with cytokine cocktail induces double negative T cells (CD3+ CD4-CD8-)

Lymphocytes were obtained from narcolepsy patients. Amplification was performed as described in example 1, but using PRDM2 peptide as the amplification antigen and stimulation. The T cell phenotype of the cultured cells was examined on day 1 and day 18. Figure 2 shows the flow cytometry results for these samples. In the results, lymphocytes were first filtered out, and then CD3+ lymphocytes were filtered out. These cells were then further analyzed for the presence of CD4 and CD 8. As can be seen from the figure, only 13% of the cells were in a double negative state after day 1. Most cells are CD4 +. After 18 days of expansion with the cytokine mixture, 92% of the cells were double negative (CD3+ CD4-CD8-) T cells.

In addition, IFN- γ production from expanded lymphocytes induced upon PRDM2 peptide stimulation was examined. As shown in FIG. 3, only 0.89% of the cells produced IFN- γ upon stimulation with PRDM2 on day 1. In contrast, the day 18 samples showed a significantly increased population of cytokine-producing PRDM 2-specific T cells. Since most of the T cells were in the CD3+ CD4-CD 8-state, this suggests that the expansion protocol results in the formation of clinically relevant double negative T cells.

Example 6-sorted dextramer cells produce IFN- γ in response to autologous EBV-LCL carrying peptide and protein antigens

The amplification protocol of example 1 was also performed using blood from two different narcolepsy patients using PRDM2 as the amplification peptide. In two different settings, each patient's DNAseB or PRDM2 peptide was added to the cytokine mixture in the culture medium. CD3+ CD4-CD8-T cells and conventional CD8+ T cells, which are MHC restricted and directed against PRDM2 or DNAseB, were sorted by flow cytometry and tested for recognition of naturally processed and presented epitopes. T cells from the same patient were loaded with peptide or recombinant protein and IFN- γ production was measured. The results of the experiments are summarized in table 1:

TABLE 1

The measurements are IFN-. gamma.concentrations in pg/ml. This experiment shows that a cytokine mixture of IL-2, IL-15 and IL-21 in combination with a specific antigen is able to expand clinically relevant T cells, in particular T cells specific for the antigen in peripheral blood. T cells may preferentially be present in the so-called DN CD3+ CD4-CD8-T cell population, and these T cells are highly functionally active, they produce IFN- γ and recognize biologically and medically relevant targets.

Example 7 analysis of lymphocytes expanded with cytokine cocktail and tumor-associated antigens

The amplification process was performed as described in example 1, except that INO80E and UCHL3 were used as the amplification antigen instead of NY-ESO-1. After 18 days of expansion, cells were stimulated with INO80E or UCHL3 and analyzed by flow cytometry. The results of flow cytometry are shown in fig. 4 and 5. Fig. 4A shows signals of cells separated by side scatter and forward scatter. The lymphocytes were gated as indicated by the black oval circle. The filtered lymphocytes were then re-filtered for determination of the presence of CD 3. CD3+ cells were further analyzed by isolation based on the presence or absence of CD8 and CD 4. In this experiment, 41% of the cells were CD8+, 29.6% were CD4+ and 26.5% were double negative. Then, the amount of IFN-. gamma.production by double negative cells and CD8+ cells was measured when stimulated with the target antigens INO80E and UCHL 3. It should be noted that T cells were measured objectively using MHC class 1/peptide complexes. According to the results shown in fig. 5A, 1.4% of CD8+ cells produced IFN- γ upon stimulation with INO80E, but 0.56% of double negative T cells also produced IFN- γ upon stimulation (see fig. 5B). As shown in fig. 5, activation of UCHL3 gave similar results, with 0.34% of CD8+ cells producing IFN- γ. 0.45% of double negative cells also produced IFN-. gamma. (see FIG. 5D).

Cells stimulated with INO80E were further analyzed for cytokine production. FIGS. 6A-6C also show gating of IFN-. gamma.producing CD8+ T cells. The analysis of cells producing CD107a, CD127(IL-7R) and CD117 is shown in FIGS. 6D-6F. The grey signal represents the entire CD3+ CD8+ T cell population (without regard to antigen specificity), and the black signal represents antigen-specific CD3+ CD8+ T cells. Analysis showed that TAA-reactive CD8+ T cells expressed CD107a, a marker associated with cytotoxicity, and CD127, an IL-7R receptor that mediates survival signaling, but did not express CD117, a marker of T cells with stem cell-like properties.

Example 8 PBMC amplification with cytokine mixture and CMVpp65 peptide

PBMCs were obtained from glioblastoma patients and expanded according to example 1 with a cytokine mixture of IL-2, IL-15 and IL-21 but in combination with CMVpp65 peptide. This experiment shows that stimulation with the CMVpp65 peptide in combination with a cytokine mixture of IL-2, IL-15 and IL-21 results in robust expansion of high affinity antigen-specific T cells (tetramer assay). The results are shown in fig. 7A to 7D. APC-CMV bzw, PE-CMV and FITC-CMV represent I) the same MHC class I HLA-0201 molecule, ii) the same peptide. But the MHC molecule is mutated. The only difference is the mutation of the MHC molecule-which detects T cells with different affinities. CMV tetramers were mutated in different ways. APC-CMV (medium affinity, mutation at position 245) is between PE-CMV (high affinity) and FITC-CMV (wild type, relatively low affinity). The data show that only mutant tetramers that allow high affinity T cell receptor binding detect high affinity T cells (as well as T cells with low and moderate affinities). It is believed that high affinity T cells mediate stronger immune effector functions and are superior in removing tumor cells and/or pathogens.

Example 9 analysis of the frequency of tumor-reactive T cells after expansion of lymphocytes from peripheral blood with cytokine mixtures and NY-ESO-1

PBMCs were obtained from pancreatic cancer patients. The experiment was performed as described in example 1. Figure 8 shows the results of flow cytometry analysis of the amplified samples at day 0 and day 18. Although IFN- γ production was only 1.85 upon NY-ESO-1 stimulation on day 1, this concentration increased dramatically to 9.25 on day 18 (see FIGS. 8B and 8D). Thus, the number of NY-ESO-1 specific T cells increased dramatically during expansion.

Example 10 expansion of survivin-reactive T cells from peripheral blood of cancer patients

PBMCs were obtained from glioblastoma patients and were amplified with a cytokine mixture of IL-2, IL-15 and IL-21 but in combination with known TAA survivin according to example 1. Cells were also analyzed using flow cytometry after 1 and 18 days of culture. The expanded cells were stimulated with survivin prior to analysis. Cells were grouped into CD4+ T cells, CD8+ T cells, and double negative T cells. In these groups, the concentrations of IL-2, IFN-. gamma.and TNF-. alpha.were determined. Fig. 9A shows the results for CD4+ cells, fig. 9B shows the results for double negative cells, and fig. 9C shows the results for CD8+ cells. The results can be summarized as follows: at time point 0(T0), no T cell response defined by cytokine production could be detected. In contrast, after 18 days of expansion, strong cytokine production was found in response to survivin stimulation, demonstrating the presence of survivin-specific T cells. In this regard, it must be noted that the T cell response is often very difficult to induce anti-survivin (indece against survivin).

Example 11 phenotypic analysis of expanded lymphocytes

PBMCs were obtained from glioblastoma patients. Amplification was performed as described in example 1. Day 0 and day 18 samples were analyzed for the presence of CD45RA and CCR7 using flow cytometry. The results are shown in fig. 10A and 10B. For this experiment, cells were grouped into the following groups. Positive signals for CD45RA and CCR7 in the upper right part of the figure represent precursor cells. The bottom right CCR7+ CD45 RA-is the central memory cell. The positive signals for CD45RA and CCR7 "in the upper left region are effector cells, and the negative signals in the two lower left regions are peripheral memory cells. As shown by comparison of fig. 10A and 10B, it is apparent that the number of central memory cells was greatly increased, i.e., from 3.72 to 21.1, due to the expansion with the cytokine mixture. The number of peripheral memory cells increases slightly, while the number of effector cells decreases dramatically. The number of precursor cells only decreased slightly from 65.4 to 57.4. Thus, expansion with the cytokine combination of the invention enriches central memory T cells.

Example 12 analysis of differentiation of different T cell subsets when expanding with cytokine mixtures of IL-2, IL-15 and IL-21 together with different TAAs

According to example 1, PBMCs from pancreatic cancer patients were treated with different antigens: the cell surface binding portion of mesothelin GPI, survivin and NY-ESO-1. The results are summarized in table 2 below:

TABLE 2

In the first column, the T cell subpopulations to be analyzed are shown: CD4+, CD8+, CD4-, and CD 8-. In the second column, an antigenic stimulus is defined. In the following columns, the percentages of central memory cells, precursor cells, differentiated effector cells and peripheral memory cells at day 0 and day 18 of the different experiments are shown. Thus, some antigens such as mesothelin drive the expansion of, inter alia, precursor cells defined by CD45RA + CCR7+ and central memory cells defined by CD45RA-CCR7 +.

Example 13 enrichment of precursor T cells and c-kit expression

PBMCs from glioblastoma patients were amplified with antigenic peptides (EGRvIII, NY-ESO-1 or survivin) according to the protocol of example 1. Cells were stimulated with the same peptide and analyzed by flow cytometry, and the results are summarized in table 3:

TABLE 3

The numbers are the percentage of T cell populations in the parental CD4+, CD8+, or double negative phenotype of each respective antigen at time point 18 days when compared to time point 0 (start of amplification). The enrichment of the subset of precursor T cells defined by CD45RA + CCR7+ associated with strong expression of antigen and c-kit (CD117) was examined. C-kit is a marker for T cells with stem cell-like properties. CD117+ T cells are associated with providing long-term memory cell populations. A strong induction of the cytotoxic/degranulation marker CD107a in stimulated T cells was also observed.

Example 14 amplification protocol Using cytokine cocktail and TAA peptides 4-1BB and TIM-3 positive T cells

PBMCs were derived from pancreatic cancer patients and amplified according to example 1. The TAA peptide is also derived from GPI, NY-ESO-1 and survivin. Flow cytometry was used to detect the presence of different markers. The markers are: 4-1BB, CD25, CD127, CTLA-4, LAG3, PD1 and TIM 3. The results are summarized in table 4:

TABLE 4

In table 4, T0 represents the concentration of each subpopulation defined in the second column 2 before expansion (T0). Ag represents 18 days of antigen stimulation and cytokine-driven expansion. The 4-1BB value and the TIM3 value indicate the antigen experienced (antigen experienced) T cells. As a result, 4-1BB + T cells were found to be significantly increased in some experiments and cell subsets. For example, in the CD4+ subset, stimulation with NY-ESO-1 resulted in a 200-fold increase (from 0.03 to 5.96) in 4-1BB + T cells.

Example 15 detailed analysis of cellular markers in lymphocytes expanded from peripheral blood of glioblastoma patients

Experiments were carried out according to the protocol of example 1 with EGVRVIII, NY-ESO-1 or survivin, respectively. The results are summarized in table 5.

TABLE 5

Lymphocytes were also isolated based on the presence of CD4 or CD8 +. The numbers in the table refer to the percentage of cells positive for each marker listed in the second column of the table after T0 or 18 days of amplification (Ag). Also for most patients, strong induction of 4-1BB + T cells was found.

Example 16 analysis of lymphocytes expanded from pancreatic cancer patients and glioblastoma patients

PBMCs were obtained from pancreatic cancer patients and glioblastoma patients. Lymphocytes were expanded according to the protocol of example 1. Lymphocyte cultures for various markers were analyzed by flow cytometry. The results are shown in table 6:

TABLE 6

In the first column, it is indicated which subset of lymphocytes is filtered out. The corresponding detection markers are listed in the second column. If no markers are listed, the ranking is based on the total number of cells, the percentage of the total number of cells with the filter marker CD3+, and the percentage of the total number of cells or double negative cells with CD4+ or CD8 +. Similarly, T0 represents the pre-amplification culture and TH represents the harvest time on day 18. The numbers indicate the percentage of cells carrying the selected marker in column 2 or 1, respectively. It must be noted that most CD4+ cells after expansion are TH1 positive. In pancreatic cancer, the number of CD4 cells increased from 22.2% to 92.1%. In glioblastoma, the results were less obvious, with CD4 cells increasing only from 9.64% to 68.1%. Similarly, there was also strong expression of CXCR3+ in CD8+ T cells after expansion, 96.8% in pancreatic cancer and 85.5% in glioblastoma. Thus, almost all CD8+ cells are CXCR3 +. Furthermore, it should be noted that terminally differentiated T cells are converted into a precursor T cell subset or a central memory T cell subset.

Figure 11 shows a flow cytometric map of an experiment used to determine TH17, TH1, TH1 and TH2 in a sample from a pancreatic cancer patient. Cells were gated first with CCR6 and then with CCR3 and CCR4, respectively.

Example 17 expansion of memory T cells specific for certain antigens without stimulation with these antigens

PBMCs from pancreatic cancer patients were expanded with the tumor-associated antigen NY-ESO-1 according to example 1. The resulting cells were stimulated for 4 hours with the following tumor-associated antigens: CMV, EBNA-3a, INO80E, mesothelin, NY-ESO-1, survivin and UCHL 3. The percentage of cytokine-producing T cells was determined using flow cytometry. Different T cell subsets are shown in table 7: CD8+, CD4+, and double negative numbers. The following cytokines were detected: IFN-gamma, IL-2 and TNF. Interestingly, not only cytokine-producing and therefore tumor-reactive T-cells were obtained by stimulation with NY-ESO-1, but also with other antigens not used in the amplification process. Similarly, stimulation with antigens of viral targets (CMV and EBNA-3a) yielded T cell-producing cytokines, indicating the presence of T cells specific for these targets.

TABLE 7

Example 18 cytotoxicity after HPV L1 specific amplification

In this example, peripheral blood was obtained from patients with severe HPV disease (HPV33 and HPV 56). Stimulation was performed with HPV L1 peptide and a cytokine mixture comprising IL-2, IL-15 and IL-21. After 18 days of expansion, cells were stimulated with L1 peptide and measured by flow cytometry. The results are shown in fig. 12. Fig. 12D to 12F show gating of lymphocyte CD3+ and CD8+, respectively. In fig. 12A, 12B and 12C, the rectangles mark the regions showing cells expressing the cytokine CD107a, the cytokine CD107a being an antigen-specific degranulation/cytotoxicity marker. Fig. 12A is the result obtained using L1 peptide, fig. 12B is a positive control, and fig. 12C has only medium as a negative control. Thus, the percentage of cells producing CD107a increased from 0.77 to 2.23.

Example 19 recognition of autologous tumor cells by T cells expanded with cytokine cocktail and tumor antigens

PBMCs from glioblastoma patients were stimulated with NY-ESO-1 peptide and CMVpp65 peptide. Peripheral blood from glioblastoma patients was treated according to the protocol of example 4, where in both settings, the stimulatory peptides were NY-ESO-1 or CMVpp 65. Also, after 18 days, cytokine production was quantitated using different stimulating antigens or autologous tumor cells. It was found that antigen-specific expansion of T cells using a mixture of peptides and cytokines leads to expansion of T cells against stimulatory targets as well as T cells against autologous tumor cells of the patient himself. This reactivity was not present until T cells were expanded with NY-ESO-1 and cytokines. The data are summarized in table 8. The numbers represent the percentage of T cells present in the parental T cell population.

TABLE 8

Example 20 comparison of lymphocyte expansion Using two different cytokine combinations and TAA stimulation

Peripheral blood was obtained from pancreatic cancer patients and treated according to the protocol of example 4. In a different experimental setup, NY-ESO-1 or survivin was used with a cytokine mixture. Furthermore, the same experiment was performed without addition of cytokines or in the combination of IL-7 and IL-2. After 18 days of amplification, the cells were assayed for IFN- γ production when stimulated by NY-ESO-1 or the survivin peptide mixture. The results are shown in FIG. 13. FIG. 13 shows that IFN- γ production and therefore antigen-specific lymphocyte concentration is greatly increased following expansion with IL-2, IL15 and IL-21 compared to other cytokine compositions or no cytokine.

Example 21-comparison of lymphocyte expansion Using two different cytokine combinations and TAA stimulation

The experiment of example 20 was repeated with the viral antigens EBNA-1, EBNA-3a and CMVpp 65. The results are summarized in fig. 14. FIG. 14 shows that IFN- γ production and therefore antigen-specific lymphocyte concentration is greatly increased following expansion with IL-2, IL15 and IL-21 compared to other cytokine compositions or no cytokine.

Example 22 analysis of Treg expansion with cytokine cocktail

PBMC-derived T cells were cultured in the presence of cytokine mixture IL-2, IL-15 and IL-21 and tregs (regulatory T cells) were identified before and after T cell expansion using flow cytometry. The results are shown in FIG. 15. Shown from left to right in fig. 15: t cells were gated with CD4+ T cells and then with CD25high, which indicates high expression of IL-2 receptor on activated T cells. Cells were then gated with IL-2R (high CD125) cells and tested for expression of IL-7 receptor (CD127) and Foxp3 (intracellular). Treg cells were defined as CD4+ CD25high, Foxp3+ and CD127- (CD 127-negative). The number of tregs was initially low (0.07% in CD4+ T cells) and even lower (0.01%) after T cell expansion. This data means that the cytokine mixture is excellent in T cell expansion, as IL-2 is known to drive strong Treg expansion, it also emphasizes the synergistic effect of the IL-2/IL-15/IL-21 combination blocking Treg expansion, while allowing anti-tumor-directed T cell expansion.

Example 23 analysis of post-amplification VB family distribution

T cells were expanded for 21 days using the cytokine cocktail IL-2/IL-15/IL-21 together with NY-ESO-1 antigen. Flow cytometry analysis was performed before (T0 ═ time point 0) and after (TH ═ time of harvest) expansion. T cells stimulated with cytokines alone served as controls. The results are summarized in table 9. It should be noted that VB7.2 family in CD8+ T cells expanded in GRex flasks and VB13.2 family in CD8+ T cells (also expanded in GRex flasks) strongly preferentially expanded. This data shows that the presence of antigen (NY-ESO-1) drives preferential amplification of the VB family of individual TCRs-and also shows the diversity of responses driven by antigen (not only cytokines). The focused but diverse TCR VB family suggests diverse anti-target directed T cell responses.

TABLE 9

TH: harvest time point, 21 days; G-Rex: G-Rex flask, NY-ESO-1 stimulus; cyto: cytokines only, no stimulation; GE: GE wave System, NY-ESO-1 stimulation

Example 24 PD1 marker in CD8+ cells

T lymphocytes were expanded using IL-2/IL-15/IL-21 and NY-ESO-1 peptide mixture. The upper diagram: prior to cytokine/TAA amplification, the following figure: after cytokine/TAA expansion T cells have been cultured in the presence of IL-2/IL-15/IL-21. Flow cytometry analysis was performed before and after T cell stimulation. From left to right: i) gated with lymphocytes, ii) gated with CD3+ T cells, iii) gated with CD4/CD 8. Upper right: PD1 expression on CD8+ activated T cells (37% T cells). cytokine/TAA expanded T cells showed reduced expression of PD1 on CD4CD8+ T cells. This data shows that cytokine/peptide expanded T cells show a reduced frequency of PD1+ T cells on CD8+ T cells-which means that these expanded T cells can show a longer life-span and thus an excellent effect against tumor cells.

Example 25 multiple antigens

A peptide mixture consisting of 12 individual peptides from the HPV 33L 1 protein was used with a cytokine mixture to stimulate T cells from patients with HPV + lesions. On day 21, T cells were tested for cytotoxicity against each individual peptide. Here, autologous EBV-transformed B cells were taken and loaded with peptides 1-12, followed by standard Cr51 assays. This assay measures the ability of T cells to kill a particular target. The results are shown in FIG. 16.

T cells from donor a strongly recognized peptide 11 and peptide 12. T cells from donor B recognized peptide 7 to peptide 12 robustly and were strongly reactive to peptide 11. These data show that expansion of T cells with IL-2/IL-15/IL-21 cytokine cocktail and peptides leads to i) generation of cytotoxic T cells, and ii) T cell responses are diverse and focused on a collection of variants of a single peptide species. This means that T cells stimulated with peptide/cytokine mixtures can be preferentially cytotoxic and they can also target several epitopes-this makes them more diverse in recognizing tumor cells or virus-infected cells that display a different set of peptide species on their surface.

Example 26 CD117 expression before and after cytokine and NY-ESO-1 driven PBMC expansion

PBMCs were expanded with IL-2/IL-15/IL-21-mix in the presence of the tumor associated antigen NY-ESO-1. Cells were analyzed by flow cytometry before (fig. 18) and after (fig. 19) expansion. First, CD3+ T cells were gated, and then CD3+ T cells were gated with CD4+ T cells and CD8+ T cells. CD117+ cells were then detected with CD8+ T cells (upper panel) and CD4+ T cells (lower panel, left). The middle graph is as follows: CD45RA/CCR7 expression in CD8+ T cells and CD4+ T cells had a high CD45RA + CCR7+ T cell population, representative of precursor T cells (middle panel). And (3) right: CD117+ T cells (blue marker) were present in effector T cells among CD8+ T cells; CD117+ T cells can be found in different T cell populations of the CD4+ T cell subpopulation. This data indicates that CD117 expression (a marker for T cell "stem cell" presence) is present in different T cell subsets. CD117+ T cells are useful in providing a source of long-term T cell memory.

First, CD3+ T cells were gated, and then CD3+ T cells were gated with CD4+ T cells and CD8+ T cells. CD8+ T cells (upper panel) and CD4+ T cells (lower panel, left) have very strong expression and a high frequency of CD117+ cells. The middle graph is as follows: CD45RA/CCR7 expression in CD8+ T cells and CD4+ T cells had a high CD45RA + CCR7+ T cell population, representative of precursor T cells (middle panel). And (3) right: CD117+ T cells (blue marker) were present in peripheral effector T cells of CD45RA + CCR7-T cells (effector cells). CD 84T cells, CD117+ T cells, can be found in the T cell precursor CD45RA + CCR7+ population. This data indicates that CD117 expression (a marker for T cell "stem cell" presence) is present in different T cell subsets. CD117+ T cells are useful in providing a source of long-term T cell memory. It was also shown that the cytokine mixture strongly expanded CD117+ T cells, which brought long-term immune cell memory, and that CD117+ T cells were located in precursor T cells-to ensure a long-term tumor immune response.

Example 27 expansion of TIL cultures from glioblastoma

This example describes the process of culturing TILs from pancreatic cancer for functional testing and immunotherapy.

See example 1 for materials, instruments and supplies.

Tumor tissue obtained from a patient is placed in a sterile container. Cutting the tissue into 1 to 2mm pieces with a sterile scalpel³The block of (1). The tissue blocks were placed into wells of 24-well plates, 1 block per well. Cell culture media was prepared using Cellgro with 10% human AB serum. To this medium, IL-2, IL-15 and IL-21 were added so that the final concentration of IL-2 was 1000u/ml and the final concentrations of IL-15 and IL-21 were each 10 ng/ml. In addition, PEST and amphotericin were added to the medium. To each well 1ml of medium was added and the cell culture was incubated at 37 ℃ for 7 days. In parallel, PBMCs from healthy donors were cultured in Cellgro containing 10% human AB serum. PBMC concentration was determined and adjusted to 10⁶And/ml. The PBMCs were then irradiated at 55Gy for 18 minutes. After irradiation, OKT3 was added to a final concentration of 10 ng/ml. This culture was termed feeder cells with OKT 3. On day 10 of TIL culture, 100 μ l of feeder cell culture with OKT3 was added to each well of a 24-well plate. Thus, the final concentration of the culture in each well was 10ng/ml OKT3 for a total of 10⁶And (4) feeding cells. The ratio of TIL to feeder cells was about 1: 10.

Example 28 amplification of TIL cultures from pancreatic cancer

Tumor tissue obtained from a patient is placed in a sterile container. Cutting the tissue into 1 to 2mm pieces with a sterile scalpel³The block of (1). The tissue blocks were placed into wells of 24-well plates, 1 block per well. Cell culture media was prepared using Cellgro with 10% human AB serum. To this medium, IL-2, IL-15 and IL-21 were added so that the final concentration of IL-2 was 1000u/ml and the final concentrations of IL-15 and IL-21 were each 10 ng/ml. In addition, PEST and amphotericin were added to the medium. To each well 1ml of medium was added and the cell culture was incubated at 37 ℃ for 4 days. In parallel withPBMCs from healthy donors were cultured in Cellgro containing 10% human AB serum. PBMC concentration was determined and adjusted to 10⁶And/ml. The PBMCs were then irradiated at 55Gy for 18 minutes. After irradiation, OKT3 was added to a final concentration of 10 ng/ml. This culture was termed feeder cells with OKT 3. On day 10 of TIL culture, 100 μ l of feeder cell culture with OKT3 was added to each well of a 24-well plate. Thus, the culture in each well contained OKT3 at a final concentration of 10ng/ml for a total of 10⁶And (4) feeding cells. The ratio of TIL to feeder cells was about 1: 10.

Example 29 Process for generating tumor cell lines from tissue

Pancreatic tumor tissue was obtained directly after surgery and placed in sterile containers. Cutting the tumor tissue into 1-2 mm pieces using a sterile scalpel³The block of (1). Each tissue mass was transferred to a well of a 24-well plate. To each well was added 1ml of medium RPMI 1640 with 10% fetal bovine serum. The medium was changed on day 7 and 14, which means that the medium was removed and replaced with fresh medium of the same kind. When tumor cells reached very high density, cultures were transferred to 6-well plates.

Example 30 determination of the correct time Point for feeder cells addition

It was found that the addition of feeder cells did not result in a good expansion of tumor infiltrating lymphocytes if the feeder cells were added at very low concentrations of lymphocytes. Fig. 20A shows lymphocyte cultures incubated for one week. Some lymphocytes are detectable, however the concentration of lymphocytes is below the baseline of expansion. Fig. 20B and 20C show the same lymphocyte cultures after two weeks of incubation. Now, the number of lymphocytes detectable therein has increased, above the baseline of expansion. Feeder cells may be added at this stage. Fig. 20D, 20E and 20E show cultures at 4 days, one week and two weeks after feeder cell addition. It can be seen in these figures that the number of lymphocytes sharply increases from the image in panel D to the image in panel F. As can be seen in panel F, lymphocytes aggregated and attacked the tumor cells in culture.

Example 31 analysis of the phenotype and activation/depletion marker expression of lymphocytes expanded from TILs obtained from glioblastomas

Tumor tissue was obtained from 16 glioblastoma patients. TIL was amplified from tissues using a cytokine mixture of IL-2, IL-15 and IL-21 according to the protocol of example 1.

A)

The expanded cells were analyzed by flow cytometry for their CD4/CD8 phenotype using antibodies against CD3, CD4, and CD 8.

The results are summarized in table 10.

Watch 10

This data shows that TIL can be expanded from each individual tumor containing predominantly CD3+ T cells. Depending on the patient, which of CD4+ T cells, CD8+ T cells, and DN T cells predominate. Most commonly, however, is CD4 +. The table also shows that cytokine cocktails are capable of expanding T cells from different CNS tumor tissues. (see column 4 "diagnosis")

B)

Expression of specific phenotype-precursor T cells (CD45RA + CCR7+), central memory T cells (CD45RA-CCR7+), peripheral memory T cells (CD45RA-CCR7-) or differentiated effector T cells (CD45RA + CCR7-) and activation/depletion markers of expanded cells among basal phenotype CD8+ T cells, CD4+ T cells and double negative T cells were analyzed by flow cytometry.

Antibodies used for flow cytometry analysis of TIL populations are summarized in table 11.

TABLE 11

The results are summarized in fig. 22A and 22B. Most CD8+ T cells, CD4+ T cells, or DN T cells are in the central memory T cell subpopulation, which has been shown to be critical for an effective anti-cancer response and long-term immune memory. The results on average show a strong expansion of DN-T cells. This subset is highly activated and carries an affinity T cell receptor. A robust expansion of the CD45RA + CCR7+ precursor T cell subset was also observed, providing a long-term memory T cell response that is favorable for long-term immune surveillance.

Expression of 4-1BB, LAG-3 or TIM-3 in T cells reported (see FIG. 3) is indicative of antigen/tumor specific T cells. This example shows that cytokine mixtures expand TIL predominantly present in a subset of memory (central memory) T cells associated with a beneficial clinical response, and also shows that TIL with markers specifically associated with antigens are expanded.

Example 32 analysis of phenotype and activation/depletion marker expression from TIL-expanded lymphocytes obtained from pancreatic cancer

Tumor tissues were obtained from 17 pancreatic cancer patients. Table 12 summarizes the age, sex, sample type and histology of the patients.

TABLE 12 cytokine mixture expansion of TILs from different pancreatic cancer tissues

Patient ID	Age (age)	Sex	Sample (I)	Histology
					Pancreatic cancer patient 1	72	M	Biopsy	Papillary adenocarcinoma
Pancreatic cancer patient 2	66	M	Tumor(s)	Ductal adenocarcinoma
					Pancreatic cancer patient 3	68	M	Tumor(s)	Adenocarcinoma of duodenum
Pancreatic cancer patient 4	67	M	Tumor(s)	Ductal adenocarcinoma
					Pancreatic cancer patient 5	81	F	Tumor(s)	Ductal adenocarcinoma
Pancreatic cancer patient 6	50	M	Biopsy	Ductal adenocarcinoma
					Pancreatic cancer patient 7	68	M	Biopsy	Ductal adenocarcinoma
Pancreatic cancer patient 8	74	F	Tumor(s)	Ductal adenocarcinoma
					Pancreatic cancer patient 9	71	M	Tumor(s)	Ductal adenocarcinoma
Pancreatic cancer patient 10	60	F	Tumor(s)	Adenocarcinoma
					Pancreatic cancer patient 11	42	F	Tumor(s)	Ductal adenocarcinoma
Pancreatic cancer patient 12	70	M	Tumor(s)	Ductal adenocarcinoma
					Pancreatic cancer patient 13	59	F	Tumor(s)	Squamous carcinoma of pancreas
Pancreatic cancer patient 14	60	F	Tumor(s)	Ductal adenocarcinoma
					Pancreatic cancer patient 15	72	M	Tumor(s)	Ductal adenocarcinoma
Pancreatic cancer patient 16	81	F	Tumor(s)	Ductal adenocarcinoma
					Pancreatic cancer patient 17	61	M	Biopsy	Ductal adenocarcinoma

TIL was amplified from tissues using a cytokine mixture of IL-2, IL-15 and IL-21 according to the protocol of example 28. The CD4/CD8 phenotype was analyzed using antibodies against CD3, CD4, and CD 8.

A)

The expanded cells were analyzed by flow cytometry for their CD4/CD8 phenotype using antibodies against CD3, CD4, and CD 8. The results are summarized in table 13.

Table 13-T cell phenotype analysis in pancreatic cancer TIL. Mainly the CD8+ TIL population.

Expanded cells in basal phenotype CD8+ and CD4+ were analyzed by flow cytometry for specific phenotype-precursor T cells (CD45RA + CCR7+), central memory T cells (CD45RA-CCR7+), peripheral memory T cells (CD45RA-CCR7-) or differentiated effector T cells (CD45RA + CCR 7-). The results are summarized in fig. 23 and 24.

Most CD8+ T cells, CD4+ T cells, or DN T cells are in the central memory T cell subpopulation, which has been shown to be critical for an effective anti-cancer response and long-term immune memory. The results averaged to show robust expansion of DN-T cells (data not shown). This subset is highly activated and carries an affinity T cell receptor. A robust expansion of the CD45RA + CCR7+ precursor T cell subset was also observed, providing a long-term memory T cell response that is favorable for long-term immune surveillance.

Expression of 4-1BB, LAG-3 or TIM-3 in T cells reported (see FIG. 24) is indicative of antigen/tumor specific T cells. This example shows that cytokine mixtures expand TIL predominantly present in a subset of memory (central memory) T cells associated with a beneficial clinical response, and also shows that TIL with markers specifically associated with antigens are expanded.

Example 33 analysis of TCR Length of T cells expanded from tumor tissue of pancreatic cancer patients

Tumor tissues were obtained from 17 pancreatic cancer patients. TIL was amplified from tissues using a cytokine mixture of IL-2, IL-15 and IL-21 according to the protocol of example 28. TCR length was measured using PCR-based methods. The "normal" image of the TCR family is a gaussian distribution of T cell receptor lengths. A single peak indicates the monoclonality of a single TCR family. The data presented in fig. 25 indicate that the TIL compositions are monoclonal or polyclonal, suggesting that focusing on autologous tumor targets, the cytokine cocktail helps to expand the focused TCR lineage.

Example 34 analysis of cytokine production in TIL-expanded lymphocytes obtained from patients with glioblastoma

Tumor tissue was obtained from a patient with glioblastoma. TIL was amplified from tumor tissue using a cytokine mixture of IL-2, IL-15 and IL-21 according to the protocol of example 1. The expanded lymphocytes are stimulated with a peptide derived from a tumor-associated antigen, EGRvrIII, NY-ESO-1 or survivin, and the percentage of cells producing any one of the cytokines IFN γ and TNF α is measured.

The results are shown in FIG. 26. For comparison, the maximum stimulation was detected with PMA/ionomycin as a positive control and the background signal was determined with medium only (negative control). The results indicate that expanded T cells recognize these common tumor antigens. Thus, the cytokine mixture expands T cells from glioblastoma that are reactive to tumor antigens that have been described as clinically relevant and relevant to clinical responses.

Example 35 analysis of cytokine production in TIL-expanded lymphocytes obtained from pancreatic cancer patients

Tumor tissue was obtained from pancreatic cancer patients.

TIL was amplified from tumor tissue using a cytokine mixture of IL-2, IL-15 and IL-21 according to the protocol of example 27. The expanded lymphocytes are stimulated with a peptide derived from a tumor-associated antigen, EGRvrIII, NY-ESO-1 or survivin, and the percentage of cells producing any one of the cytokines IFN γ and TNF α is measured. The results are shown in FIG. 27. FIG. 27 shows images of flow cytometry analysis of NY-ESO-1. The results confirmed that expanded T cells recognized these common tumor antigens. Thus, the cytokine mixture expands T cells from pancreatic tumors that are reactive to tumor antigens that have been described as clinically relevant and relevant to clinical responses.

Example 36 analysis of cytokine production in TIL-expanded lymphocytes obtained from glioblastoma patients under autologous stimulation

Tumor tissue was obtained from a patient with glioblastoma. TIL was amplified from tumor tissue using a cytokine mixture of IL-2, IL-15 and IL-21 according to the protocol of example 27. The expanded lymphocytes are stimulated with autologous tumor cells. The results are shown in FIG. 28. Figure 28 shows an image of flow cytometry analysis. The results confirmed that the expanded T cells recognized autologous tumor cells. Thus, the cytokine mixture expands T cells from glioblastoma, which are reactive to autologous tumor cells, corresponding to the patient's own mutations.

Example 37: association of TCR use in pancreatic cancer with recognition of autologous tumor cells

Tumor tissue was obtained from a patient with glioblastoma. TIL was amplified from tumor tissue using a cytokine mixture of IL-2, IL-15 and IL-21 according to the protocol of example 27. The expanded lymphocytes are stimulated with autologous tumor cells. TILs were gated first with CD3+ T cells, and then with CD4+ T cells and CD8+ T cells. The frequency of individual V β families was detected using a panel of TCR VB antibodies. This TCR panel covers about 75% of the human TCR lineage, and thus may not capture all TCR v β family members sufficiently. The TCR V β family distribution is about 2 to 6% in each family, except for TCR VB2 which can reach more than 10%. Table 14 shows preferential expansion of V.beta.2 family in TILs from glioblastoma patients.

TABLE 14

The data in table 14 indicate that a single TCR VB is preferentially amplified in TIL. It should be noted that clonality can only be resolved by sequencing. It should be noted that cytokine cocktails amplify different TCRV β families in each patient. This is true for glioblastoma patients as well as pancreatic cancer patients. Also note that some TILs consist of single or two VB families, showing highly focused TCR VB expansion, suggesting an antigen-driven T cell expansion process. Some TILs that we have demonstrated as preferentially amplifying appear to be monoclonal. Thus, the cytokine cocktail of IL-2, IL-15, and IL-21 amplifies a focused T cell response against the patient's own tumor cells.

Example 38 correlation of the use of TCR in pancreatic cancer with the identification of autologous tumor cells

Tumor tissue was obtained from pancreatic cancer patients. TIL was amplified from tumor tissue using a cytokine mixture of IL-2, IL-15 and IL-21 according to the protocol of example 28.

Cells were stained by flow cytometry using CD3, CD4, and CD8 in combination with TCR V β antibodies. This group covers up to 75% of the entire TCR VB lineage. Certain T cell family members are not captured in the group if they are present in 25% of the uncovered group. The results are summarized in table 15.

Watch 15

IFN γ production was measured 3 days after exposing TILs to autologous tumor cells (single cell suspension). High levels of IFN γ production were observed after autologous tumor cells only (see fig. 30). IFN γ production was completely blocked with an anti-W6/32 antibody (blocking CD8+ TIL). As a control, antibody L243 blocking CD4+ TIL failed to block reactivity, and reactive T cells were CD8+ cells in all expanded lymphocytes. The data show that cytokine mixtures expand TILs that are very focused and specifically recognize autologous tumor cells from pancreatic cancer patients. Monoclonal TCR family in TILs from pancreatic cancer patients: IL-2, IL-15 and IL-21 preferentially amplified a single TCR VB family, and this preferential amplification of the T cell family is associated with antigen specificity and focused anti-tumor reactivity. Some of these preferentially expanded VB families contain monoclonal T cells defined by a single TCR VB chain. This monoclonal TCR expansion indicates a focused anti-cancer response. Functional response analysis showed that the monoclonally expanded TILs recognized autologous tumor cells.

Example 39 analysis of cytolytic response of expanded TILs from glioblastoma patients to autologous tumor cells

Tumor tissue was obtained from a patient with glioblastoma. TIL was amplified from tumor tissue using a cytokine mixture of IL-2, IL-15 and IL-21 according to the protocol of example 28. Autologous tumor cells were generated according to the protocol of example 29. Autologous tumor cells were radiolabeled and incubated with expanded TIL for 4 hours. Killing of tumor cells results in the release of radioactivity, which is then measured. The principle of this method is shown in fig. 21. The results are shown in fig. 31, which demonstrates the cytotoxic effect depending on the ratio of TIL to tumor cells. The data show that cytokine mixtures expand TILs that are strongly cytotoxic to autologous tumor cells.

Alternatively, the cytotoxicity of expanded monoclonal T cells and/or preferentially expanded TIL may be tested in the same assay. The results are shown in FIG. 32. Table 16 shows that these immune responses are specific for autologous tumor cells. The data show that cytokine cocktails expand TILs with specific reactivity against autologous tumors, including cytotoxic reactions.

B)

In parallel experiments, autologous tumor cells were incubated with TIL expanded from glioblastoma with a cytokine mixture of IL-15, IL-21 and IL-2 for three days. After incubation, IFN γ production (pg/mL) was measured by ELISA. To identify whether CD4+ cells or CD8+ cells are responsible for the tumor response, antibodies were used that blocked MHC class I antigen responses (W6/32) affecting CD8+ T cells or blocked HLA-DR (MHC class II responses) affecting CD4+ T cells. The results are summarized in table 16.

TABLE 16

TILs showing the absence of IFN γ production were strongly cytotoxic as measured in a standard CR51 release assay. The data show that TIL produces IFN γ against autologous tumor cells in a specific manner.

Example 40 analysis of cytolytic response of TIL expanded from pancreatic cancer patients to autologous tumor cells

Tumor tissue was obtained from pancreatic cancer patients. TIL was amplified from tumor tissue using a cytokine mixture of IL-2, IL-15 and IL-21 according to the protocol of example 28. Autologous tumor cells were generated according to the protocol of example 29. Autologous tumor cells were radiolabeled and incubated with expanded TIL for 4 hours. Killing of tumor cells results in the release of radioactivity, which is then measured. The principle of this method is shown in fig. 7. For comparison and as a control, an autologous (melanoma) -TIL system was used. The results are shown in fig. 33, which demonstrates the cytotoxic effect. The data show that cytokine cocktails also amplify TIL from pancreatic cancer patients. These TILs are highly focused-based on TCR use-and show specific reactivity (including cytotoxic reactions) against autologous tumors.

Example 41-analysis of CXCR3 expression of CD4+ cell distribution

Tumor tissue was obtained from a patient with glioblastoma. TIL was amplified from tumor tissue using a cytokine mixture of IL-2, IL-15 and IL-21 according to the protocol of example 27. Cells were analyzed by flow cytometry for expression of markers that define T cell function and homing.

The analytical results are summarized in table 17. Prior to expansion, Th1 cells and CXCR3 were less than 10%. The data show that the cytokine mixture yields a T cell product in which the CD8+ cells consist essentially of CXCR3+ CD8+ T cells. This phenotype allows entry into the tumor tissue. CD4+ T cells almost all have T_H1Property (profile). T is_H1The properties (IFN γ and TNF α production) result in an improved anti-tumor response. There are also CD3+ CD4-CD8- (DN) T cells, a subset of T cells, associated with strong autoimmune and tumor responses, which also express the marker CXCR3, which is better able to penetrate into tumor tissues.

TABLE 17

Example 42 identification of shared tumor antigens by TILs from pancreatic cancer patients

TAA as 15 overlapping peptides were incubated with TIL for 3 days and IFN γ production (pg/mL) was measured by ELISA. Furthermore, the antigen response was blocked by antibodies that block the MHC class I antigen response (W6/32) affecting CD8+ T cells or block HLA-DR (MHC class II response, L243) affecting CD4+ T cells. The IFN γ production (pg/mL) in each sample is shown in Table 18. The IFN γ product in TIL is antigen specific as demonstrated by blocking with anti-MHC class I (blocking CD8+ T cells) or with anti-MHC class II (blocking CD4+ T cells). The results demonstrate that TIL produces IFN γ against consensus cancer antigens, particularly NY-ESO-1 or mesothelin from pancreatic cancer patients.

Watch 18

Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

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Sequence listing

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Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

20 25 30

Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile

35 40 45

Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe

50 55 60

Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu

65 70 75 80

Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

85 90 95

Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile

100 105 110

Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala

115 120 125

Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe

130 135 140

Cys Gln Ser Ile Ile Ser Thr Leu Thr

145 150

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Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr

1 5 10 15

Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His

20 25 30

Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala

35 40 45

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

50 55 60

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

65 70 75 80

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

85 90 95

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

100 105 110

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

115 120 125

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

130 135 140

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

145 150 155 160

Thr Ser

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Met Glu Arg Ile Val Ile Cys Leu Met Val Ile Phe Leu Gly Thr Leu

1 5 10 15

Val His Lys Ser Ser Ser Gln Gly Gln Asp Arg His Met Ile Arg Met

20 25 30

Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp

35 40 45

Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys

50 55 60

Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala

65 70 75 80

Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu

85 90 95

Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg

100 105 110

Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu

115 120 125

Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His

130 135 140

Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser

145 150 155

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Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp

1 5 10 15

Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly

20 25 30

Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala

35 40 45

Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly Pro

50 55 60

His Gly Gly Ala Ala Ser Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala

65 70 75 80

Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe

85 90 95

Ala Thr Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp

100 105 110

Ala Pro Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val

115 120 125

Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln

130 135 140

Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met

145 150 155 160

Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser

165 170 175

Gly Gln Arg Arg

180

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Met Gly Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp

1 5 10 15

His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Cys Ala

20 25 30

Cys Thr Pro Glu Arg Met Ala Glu Ala Gly Phe Ile His Cys Pro Thr

35 40 45

Glu Asn Glu Pro Asp Leu Ala Gln Cys Phe Phe Cys Phe Lys Glu Leu

50 55 60

Glu Gly Trp Glu Pro Asp Asp Asp Pro Ile Glu Glu His Lys Lys His

65 70 75 80

Ser Ser Gly Cys Ala Phe Leu Ser Val Lys Lys Gln Phe Glu Glu Leu

85 90 95

Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys Asn Lys

100 105 110

Ile Ala Lys Glu Thr Asn Asn Lys Lys Lys Glu Phe Glu Glu Thr Ala

115 120 125

Lys Lys Val Arg Arg Ala Ile Glu Gln Leu Ala Ala Met Asp

130 135 140

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Met Ala Leu Pro Thr Ala Arg Pro Leu Leu Gly Ser Cys Gly Thr Pro

1 5 10 15

Ala Leu Gly Ser Leu Leu Phe Leu Leu Phe Ser Leu Gly Trp Val Gln

20 25 30

Pro Ser Arg Thr Leu Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu

35 40 45

Asp Gly Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser Pro Arg

50 55 60

Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr Glu

65 70 75 80

Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys Asn Val Lys Leu

85 90 95

Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro Pro

100 105 110

Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu Leu Leu Phe Leu Asn Pro

115 120 125

Asp Ala Phe Ser Gly Pro Gln Ala Cys Thr Arg Phe Phe Ser Arg Ile

130 135 140

Thr Lys Ala Asn Val Asp Leu Leu Pro Arg Gly Ala Pro Glu Arg Gln

145 150 155 160

Arg Leu Leu Pro Ala Ala Leu Ala Cys Trp Gly Val Arg Gly Ser Leu

165 170 175

Leu Ser Glu Ala Asp Val Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu

180 185 190

Pro Gly Arg Phe Val Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu

195 200 205

Val Ser Cys Pro Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg

210 215 220

Ala Ala Leu Gln Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp

225 230 235 240

Ser Val Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Val Leu Gly

245 250 255

Gln Pro Ile Ile Arg Ser Ile Pro Gln Gly Ile Val Ala Ala Trp Arg

260 265 270

Gln Arg Ser Ser Arg Asp Pro Ser Trp Arg Gln Pro Glu Arg Thr Ile

275 280 285

Leu Arg Pro Arg Phe Arg Arg Glu Val Glu Lys Thr Ala Cys Pro Ser

290 295 300

Gly Lys Lys Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys

305 310 315 320

Trp Glu Leu Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met

325 330 335

Asp Arg Val Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu

340 345 350

Lys His Lys Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro Glu Ser Val

355 360 365

Ile Gln His Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp Ile

370 375 380

Arg Lys Trp Asn Val Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu

385 390 395 400

Val Asn Lys Gly His Glu Met Ser Pro Gln Ala Pro Arg Arg Pro Leu

405 410 415

Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val Lys Gly Arg Gly Gln

420 425 430

Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr Pro Gly Tyr

435 440 445

Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val Pro Pro Ser Ser

450 455 460

Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr Cys Asp Pro Arg Gln

465 470 475 480

Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gln Asn Met Asn

485 490 495

Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe Leu Gly Gly Ala Pro

500 505 510

Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn Val Ser Met Asp Leu

515 520 525

Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val Leu Pro Leu Thr Val

530 535 540

Ala Glu Val Gln Lys Leu Leu Gly Pro His Val Glu Gly Leu Lys Ala

545 550 555 560

Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile Leu Arg Gln Arg Gln

565 570 575

Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln Gly Gly Ile Pro Asn

580 585 590

Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu Ala Leu Ser Gly Thr

595 600 605

Pro Cys Leu Leu Gly Pro Gly Pro Val Leu Thr Val Leu Ala Leu Leu

610 615 620

Leu Ala Ser Thr Leu Ala

625 630

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Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala

1 5 10 15

Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln

20 25 30

Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe

35 40 45

Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn

50 55 60

Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys

65 70 75 80

Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val

85 90 95

Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr

100 105 110

Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn

115 120 125

Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu

130 135 140

His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu

145 150 155 160

Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met

165 170 175

Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro

180 185 190

Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln

195 200 205

Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg

210 215 220

Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys

225 230 235 240

Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp

245 250 255

Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro

260 265 270

Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly

275 280 285

Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His

290 295 300

Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu

305 310 315 320

Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val

325 330 335

Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn

340 345 350

Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp

355 360 365

Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr

370 375 380

Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu

385 390 395 400

Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp

405 410 415

Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln

420 425 430

His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu

435 440 445

Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser

450 455 460

Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu

465 470 475 480

Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu

485 490 495

Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro

500 505 510

Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn

515 520 525

Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly

530 535 540

Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro

545 550 555 560

Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro

565 570 575

Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val

580 585 590

Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp

595 600 605

Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys

610 615 620

Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly

625 630 635 640

Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu

645 650 655

Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His

660 665 670

Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu

675 680 685

Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu

690 695 700

Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser

705 710 715 720

Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu

725 730 735

Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser

740 745 750

Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser

755 760 765

Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser

770 775 780

Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp

785 790 795 800

Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn

805 810 815

Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg

820 825 830

Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro

835 840 845

Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala

850 855 860

Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp

865 870 875 880

Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp

885 890 895

Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser

900 905 910

Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu

915 920 925

Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr

930 935 940

Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys

945 950 955 960

Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln

965 970 975

Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro

980 985 990

Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp

995 1000 1005

Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe

1010 1015 1020

Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu

1025 1030 1035

Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn

1040 1045 1050

Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg

1055 1060 1065

Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp

1070 1075 1080

Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro

1085 1090 1095

Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln

1100 1105 1110

Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro

1115 1120 1125

His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln

1130 1135 1140

Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala

1145 1150 1155

Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln

1160 1165 1170

Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys

1175 1180 1185

Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln

1190 1195 1200

Ser Ser Glu Phe Ile Gly Ala

1205 1210

<210> 8

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<213> Intelligent people

<400> 8

Met Glu Ser Arg Gly Arg Arg Cys Pro Glu Met Ile Ser Val Leu Gly

1 5 10 15

Pro Ile Ser Gly His Val Leu Lys Ala Val Phe Ser Arg Gly Asp Thr

20 25 30

Pro Val Leu Pro His Glu Thr Arg Leu Leu Gln Thr Gly Ile His Val

35 40 45

Arg Val Ser Gln Pro Ser Leu Ile Leu Val Ser Gln Tyr Thr Pro Asp

50 55 60

Ser Thr Pro Cys His Arg Gly Asp Asn Gln Leu Gln Val Gln His Thr

65 70 75 80

Tyr Phe Thr Gly Ser Glu Val Glu Asn Val Ser Val Asn Val His Asn

85 90 95

Pro Thr Gly Arg Ser Ile Cys Pro Ser Gln Glu Pro Met Ser Ile Tyr

100 105 110

Val Tyr Ala Leu Pro Leu Lys Met Leu Asn Ile Pro Ser Ile Asn Val

115 120 125

His His Tyr Pro Ser Ala Ala Glu Arg Lys His Arg His Leu Pro Val

130 135 140

Ala Asp Ala Val Ile His Ala Ser Gly Lys Gln Met Trp Gln Ala Arg

145 150 155 160

Leu Thr Val Ser Gly Leu Ala Trp Thr Arg Gln Gln Asn Gln Trp Lys

165 170 175

Glu Pro Asp Val Tyr Tyr Thr Ser Ala Phe Val Phe Pro Thr Lys Asp

180 185 190

Val Ala Leu Arg His Val Val Cys Ala His Glu Leu Val Cys Ser Met

195 200 205

Glu Asn Thr Arg Ala Thr Lys Met Gln Val Ile Gly Asp Gln Tyr Val

210 215 220

Lys Val Tyr Leu Glu Ser Phe Cys Glu Asp Val Pro Ser Gly Lys Leu

225 230 235 240

Phe Met His Val Thr Leu Gly Ser Asp Val Glu Glu Asp Leu Thr Met

245 250 255

Thr Arg Asn Pro Gln Pro Phe Met Arg Pro His Glu Arg Asn Gly Phe

260 265 270

Thr Val Leu Cys Pro Lys Asn Met Ile Ile Lys Pro Gly Lys Ile Ser

275 280 285

His Ile Met Leu Asp Val Ala Phe Thr Ser His Glu His Phe Gly Leu

290 295 300

Leu Cys Pro Lys Ser Ile Pro Gly Leu Ser Ile Ser Gly Asn Leu Leu

305 310 315 320

Met Asn Gly Gln Gln Ile Phe Leu Glu Val Gln Ala Ile Arg Glu Thr

325 330 335

Val Glu Leu Arg Gln Tyr Asp Pro Val Ala Ala Leu Phe Phe Phe Asp

340 345 350

Ile Asp Leu Leu Leu Gln Arg Gly Pro Gln Tyr Ser Glu His Pro Thr

355 360 365

Phe Thr Ser Gln Tyr Arg Ile Gln Gly Lys Leu Glu Tyr Arg His Thr

370 375 380

Trp Asp Arg His Asp Glu Gly Ala Ala Gln Gly Asp Asp Asp Val Trp

385 390 395 400

Thr Ser Gly Ser Asp Ser Asp Glu Glu Leu Val Thr Thr Glu Arg Lys

405 410 415

Thr Pro Arg Val Thr Gly Gly Gly Ala Met Ala Gly Ala Ser Thr Ser

420 425 430

Ala Gly Arg Lys Arg Lys Ser Ala Ser Ser Ala Thr Ala Cys Thr Ser

435 440 445

Gly Val Met Thr Arg Gly Arg Leu Lys Ala Glu Ser Thr Val Ala Pro

450 455 460

Glu Glu Asp Thr Asp Glu Asp Ser Asp Asn Glu Ile His Asn Pro Ala

465 470 475 480

Val Phe Thr Trp Pro Pro Trp Gln Ala Gly Ile Leu Ala Arg Asn Leu

485 490 495

Val Pro Met Val Ala Thr Val Gln Gly Gln Asn Leu Lys Tyr

500 505 510

<210> 9

<211> 944

<212> PRT

<213> Intelligent people

<400> 9

Met Asp Lys Asp Arg Pro Gly Pro Pro Ala Leu Asp Asp Asn Met Glu

1 5 10 15

Glu Glu Val Pro Ser Thr Ser Val Val Gln Glu Gln Val Ser Ala Gly

20 25 30

Asp Trp Glu Asn Val Leu Ile Glu Leu Ser Asp Ser Ser Ser Glu Lys

35 40 45

Glu Ala Glu Asp Ala His Leu Glu Pro Ala Gln Lys Gly Thr Lys Arg

50 55 60

Lys Arg Val Asp His Asp Ala Gly Gly Ser Ala Pro Ala Arg Pro Met

65 70 75 80

Leu Pro Pro Gln Pro Asp Leu Pro Gly Arg Glu Ala Ile Leu Arg Arg

85 90 95

Phe Pro Leu Asp Leu Arg Thr Leu Leu Gln Ala Ile Gly Ala Ala Ala

100 105 110

Thr Arg Ile Asp Thr Arg Ala Ile Asp Gln Phe Phe Gly Ser Gln Ile

115 120 125

Ser Asn Thr Glu Met Tyr Ile Met Tyr Ala Met Ala Ile Arg Gln Ala

130 135 140

Ile Arg Asp Arg Arg Arg Asn Pro Ala Ser Arg Arg Asp Gln Ala Lys

145 150 155 160

Trp Arg Leu Gln Thr Leu Ala Ala Gly Trp Pro Met Gly Tyr Gln Ala

165 170 175

Tyr Ser Ser Trp Met Tyr Ser Tyr Thr Asp His Gln Thr Thr Pro Thr

180 185 190

Phe Val His Leu Gln Ala Thr Leu Gly Cys Thr Gly Gly Arg Arg Cys

195 200 205

His Val Thr Phe Ser Ala Gly Thr Phe Lys Leu Pro Arg Cys Thr Pro

210 215 220

Gly Asp Arg Gln Trp Leu Tyr Val Gln Ser Ser Val Gly Asn Ile Val

225 230 235 240

Gln Ser Cys Asn Pro Arg Tyr Ser Ile Phe Phe Asp Tyr Met Ala Ile

245 250 255

His Arg Ser Leu Thr Lys Ile Trp Glu Glu Val Leu Thr Pro Asp Gln

260 265 270

Arg Val Ser Phe Met Glu Phe Leu Gly Phe Leu Gln Arg Thr Asp Leu

275 280 285

Ser Tyr Ile Lys Ser Phe Val Ser Asp Ala Leu Gly Thr Thr Ser Ile

290 295 300

Gln Thr Pro Trp Ile Asp Asp Asn Pro Ser Thr Glu Thr Ala Gln Ala

305 310 315 320

Trp Asn Ala Gly Phe Leu Arg Gly Arg Ala Tyr Gly Ile Asp Leu Leu

325 330 335

Arg Thr Glu Gly Glu His Val Glu Gly Ala Thr Gly Glu Thr Arg Glu

340 345 350

Glu Ser Glu Asp Thr Glu Ser Asp Gly Asp Asp Glu Asp Leu Pro Cys

355 360 365

Ile Val Ser Arg Gly Gly Pro Lys Val Lys Arg Pro Pro Ile Phe Ile

370 375 380

Arg Arg Leu His Arg Leu Leu Leu Met Arg Ala Gly Lys Arg Thr Glu

385 390 395 400

Gln Gly Lys Glu Val Leu Glu Lys Ala Arg Gly Ser Thr Tyr Gly Thr

405 410 415

Pro Arg Pro Pro Val Pro Lys Pro Arg Pro Glu Val Pro Gln Ser Asp

420 425 430

Glu Thr Ala Thr Ser His Gly Ser Ala Gln Val Pro Glu Pro Pro Thr

435 440 445

Ile His Leu Ala Ala Gln Gly Met Ala Tyr Pro Leu His Glu Gln His

450 455 460

Gly Met Ala Pro Cys Pro Val Ala Gln Ala Pro Pro Thr Pro Leu Pro

465 470 475 480

Pro Val Ser Pro Gly Asp Gln Leu Pro Gly Val Phe Ser Asp Gly Arg

485 490 495

Val Ala Cys Ala Pro Val Pro Ala Pro Ala Gly Pro Ile Val Arg Pro

500 505 510

Trp Glu Pro Ser Leu Thr Gln Ala Ala Gly Gln Ala Phe Ala Pro Val

515 520 525

Arg Pro Gln His Met Pro Val Glu Pro Val Pro Val Pro Thr Val Ala

530 535 540

Leu Glu Arg Pro Val Tyr Pro Lys Pro Val Arg Pro Ala Pro Pro Lys

545 550 555 560

Ile Ala Met Gln Gly Pro Gly Glu Thr Ser Gly Ile Arg Arg Ala Arg

565 570 575

Glu Arg Trp Arg Pro Ala Pro Trp Thr Pro Asn Pro Pro Arg Ser Pro

580 585 590

Ser Gln Met Ser Val Arg Asp Arg Leu Ala Arg Leu Arg Ala Glu Ala

595 600 605

Gln Val Lys Gln Ala Ser Val Glu Val Gln Pro Pro Gln Leu Thr Gln

610 615 620

Val Ser Pro Gln Gln Pro Met Glu Gly Pro Leu Val Pro Glu Gln Gln

625 630 635 640

Met Phe Pro Gly Ala Pro Phe Ser Gln Val Ala Asp Val Val Arg Ala

645 650 655

Pro Gly Val Pro Ala Met Gln Pro Gln Tyr Phe Asp Leu Pro Leu Ile

660 665 670

Gln Pro Ile Ser Gln Gly Ala Pro Val Ala Pro Leu Arg Ala Ser Met

675 680 685

Gly Pro Val Pro Pro Val Pro Ala Thr Gln Pro Gln Tyr Phe Asp Ile

690 695 700

Pro Leu Thr Glu Pro Ile Asn Gln Gly Ala Ser Ala Ala His Phe Leu

705 710 715 720

Pro Gln Gln Pro Met Glu Gly Pro Leu Val Pro Glu Gln Trp Met Phe

725 730 735

Pro Gly Ala Ala Leu Ser Gln Ser Val Arg Pro Gly Val Ala Gln Ser

740 745 750

Gln Tyr Phe Asp Leu Pro Leu Thr Gln Pro Ile Asn His Gly Ala Pro

755 760 765

Ala Ala His Phe Leu His Gln Pro Pro Met Glu Gly Pro Trp Val Pro

770 775 780

Glu Gln Trp Met Phe Gln Gly Ala Pro Pro Ser Gln Gly Thr Asp Val

785 790 795 800

Val Gln His Gln Leu Asp Ala Leu Gly Tyr Thr Leu His Gly Leu Asn

805 810 815

His Pro Gly Val Pro Val Ser Pro Ala Val Asn Gln Tyr His Leu Ser

820 825 830

Gln Ala Ala Phe Gly Leu Pro Ile Asp Glu Asp Glu Ser Gly Glu Gly

835 840 845

Ser Asp Thr Ser Glu Pro Cys Glu Ala Leu Asp Leu Ser Ile His Gly

850 855 860

Arg Pro Cys Pro Gln Ala Pro Glu Trp Pro Val Gln Glu Glu Gly Gly

865 870 875 880

Gln Asp Ala Thr Glu Val Leu Asp Leu Ser Ile His Gly Arg Pro Arg

885 890 895

Pro Arg Thr Pro Glu Trp Pro Val Gln Gly Glu Gly Gly Gln Asn Val

900 905 910

Thr Gly Pro Glu Thr Arg Arg Val Val Val Ser Ala Val Val His Met

915 920 925

Cys Gln Asp Asp Glu Phe Pro Asp Leu Gln Asp Pro Pro Asp Glu Ala

930 935 940

<210> 10

<211> 641

<212> PRT

<213> Intelligent people

<400> 10

Met Ser Asp Glu Gly Pro Gly Thr Gly Pro Gly Asn Gly Leu Gly Glu

1 5 10 15

Lys Gly Asp Thr Ser Gly Pro Glu Gly Ser Gly Gly Ser Gly Pro Gln

20 25 30

Arg Arg Gly Gly Asp Asn His Gly Arg Gly Arg Gly Arg Gly Arg Gly

35 40 45

Arg Gly Gly Gly Arg Pro Gly Ala Pro Gly Gly Ser Gly Ser Gly Pro

50 55 60

Arg His Arg Asp Gly Val Arg Arg Pro Gln Lys Arg Pro Ser Cys Ile

65 70 75 80

Gly Cys Lys Gly Thr His Gly Gly Thr Gly Ala Gly Ala Gly Ala Gly

85 90 95

Gly Ala Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly

100 105 110

Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly

115 120 125

Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala

130 135 140

Gly Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly Ala Gly

145 150 155 160

Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly

165 170 175

Ala Gly Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly

180 185 190

Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly Gly Ala Gly

195 200 205

Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala

210 215 220

Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala

225 230 235 240

Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly

245 250 255

Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly

260 265 270

Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly

275 280 285

Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly

290 295 300

Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly

305 310 315 320

Gly Ala Gly Ala Gly Gly Gly Gly Arg Gly Arg Gly Gly Ser Gly Gly

325 330 335

Arg Gly Arg Gly Gly Ser Gly Gly Arg Gly Arg Gly Gly Ser Gly Gly

340 345 350

Arg Arg Gly Arg Gly Arg Glu Arg Ala Arg Gly Gly Ser Arg Glu Arg

355 360 365

Ala Arg Gly Arg Gly Arg Gly Arg Gly Glu Lys Arg Pro Arg Ser Pro

370 375 380

Ser Ser Gln Ser Ser Ser Ser Gly Ser Pro Pro Arg Arg Pro Pro Pro

385 390 395 400

Gly Arg Arg Pro Phe Phe His Pro Val Gly Glu Ala Asp Tyr Phe Glu

405 410 415

Tyr His Gln Glu Gly Gly Pro Asp Gly Glu Pro Asp Val Pro Pro Gly

420 425 430

Ala Ile Glu Gln Gly Pro Ala Asp Asp Pro Gly Glu Gly Pro Ser Thr

435 440 445

Gly Pro Arg Gly Gln Gly Asp Gly Gly Arg Arg Lys Lys Gly Gly Trp

450 455 460

Phe Gly Lys His Arg Gly Gln Gly Gly Ser Asn Pro Lys Phe Glu Asn

465 470 475 480

Ile Ala Glu Gly Leu Arg Ala Leu Leu Ala Arg Ser His Val Glu Arg

485 490 495

Thr Thr Asp Glu Gly Thr Trp Val Ala Gly Val Phe Val Tyr Gly Gly

500 505 510

Ser Lys Thr Ser Leu Tyr Asn Leu Arg Arg Gly Thr Ala Leu Ala Ile

515 520 525

Pro Gln Cys Arg Leu Thr Pro Leu Ser Arg Leu Pro Phe Gly Met Ala

530 535 540

Pro Gly Pro Gly Pro Gln Pro Gly Pro Leu Arg Glu Ser Ile Val Cys

545 550 555 560

Tyr Phe Met Val Phe Leu Gln Thr His Ile Phe Ala Glu Val Leu Lys

565 570 575

Asp Ala Ile Lys Asp Leu Val Met Thr Lys Pro Ala Pro Thr Cys Asn

580 585 590

Ile Arg Val Thr Val Cys Ser Phe Asp Asp Gly Val Asp Leu Pro Pro

595 600 605

Trp Phe Pro Pro Met Val Glu Gly Ala Ala Ala Glu Gly Asp Asp Gly

610 615 620

Asp Asp Gly Asp Glu Gly Gly Asp Gly Asp Glu Gly Glu Glu Gly Gln

625 630 635 640

Glu

<210> 11

<211> 505

<212> PRT

<213> Intelligent people

<400> 11

Met Ser Leu Trp Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val

1 5 10 15

Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn

20 25 30

Ile Tyr Tyr His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro

35 40 45

Tyr Phe Pro Ile Lys Lys Pro Asn Asn Asn Lys Ile Leu Val Pro Lys

50 55 60

Val Ser Gly Leu Gln Tyr Arg Val Phe Arg Ile His Leu Pro Asp Pro

65 70 75 80

Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln

85 90 95

Arg Leu Val Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gln Pro

100 105 110

Leu Gly Val Gly Ile Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp

115 120 125

Thr Glu Asn Ala Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg

130 135 140

Glu Cys Ile Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly

145 150 155 160

Cys Lys Pro Pro Ile Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr

165 170 175

Asn Val Ala Val Asn Pro Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn

180 185 190

Thr Val Ile Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met

195 200 205

Asp Phe Thr Thr Leu Gln Ala Asn Lys Ser Glu Val Pro Leu Asp Ile

210 215 220

Cys Thr Ser Ile Cys Lys Tyr Pro Asp Tyr Ile Lys Met Val Ser Glu

225 230 235 240

Pro Tyr Gly Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe

245 250 255

Val Arg His Leu Phe Asn Arg Ala Gly Ala Val Gly Glu Asn Val Pro

260 265 270

Asp Asp Leu Tyr Ile Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser

275 280 285

Ser Asn Tyr Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala

290 295 300

Gln Ile Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn

305 310 315 320

Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr

325 330 335

Thr Arg Ser Thr Asn Met Ser Leu Cys Ala Ala Ile Ser Thr Ser Glu

340 345 350

Thr Thr Tyr Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu

355 360 365

Glu Tyr Asp Leu Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr

370 375 380

Ala Asp Val Met Thr Tyr Ile His Ser Met Asn Ser Thr Ile Leu Glu

385 390 395 400

Asp Trp Asn Phe Gly Leu Gln Pro Pro Pro Gly Gly Thr Leu Glu Asp

405 410 415

Thr Tyr Arg Phe Val Thr Ser Gln Ala Ile Ala Cys Gln Lys His Thr

420 425 430

Pro Pro Ala Pro Lys Glu Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu

435 440 445

Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu

450 455 460

Gly Arg Lys Phe Leu Leu Gln Ala Gly Leu Lys Ala Lys Pro Lys Phe

465 470 475 480

Thr Leu Gly Lys Arg Lys Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr

485 490 495

Thr Ala Lys Arg Lys Lys Arg Lys Leu

500 505

<210> 12

<211> 1718

<212> PRT

<213> Intelligent people

<400> 12

Met Asn Gln Asn Thr Thr Glu Pro Val Ala Ala Thr Glu Thr Leu Ala

1 5 10 15

Glu Val Pro Glu His Val Leu Arg Gly Leu Pro Glu Glu Val Arg Leu

20 25 30

Phe Pro Ser Ala Val Asp Lys Thr Arg Ile Gly Val Trp Ala Thr Lys

35 40 45

Pro Ile Leu Lys Gly Lys Lys Phe Gly Pro Phe Val Gly Asp Lys Lys

50 55 60

Lys Arg Ser Gln Val Lys Asn Asn Val Tyr Met Trp Glu Val Tyr Tyr

65 70 75 80

Pro Asn Leu Gly Trp Met Cys Ile Asp Ala Thr Asp Pro Glu Lys Gly

85 90 95

Asn Trp Leu Arg Tyr Val Asn Trp Ala Cys Ser Gly Glu Glu Gln Asn

100 105 110

Leu Phe Pro Leu Glu Ile Asn Arg Ala Ile Tyr Tyr Lys Thr Leu Lys

115 120 125

Pro Ile Ala Pro Gly Glu Glu Leu Leu Val Trp Tyr Asn Gly Glu Asp

130 135 140

Asn Pro Glu Ile Ala Ala Ala Ile Glu Glu Glu Arg Ala Ser Ala Arg

145 150 155 160

Ser Lys Arg Ser Ser Pro Lys Ser Arg Lys Gly Lys Lys Lys Ser Gln

165 170 175

Glu Asn Lys Asn Lys Gly Asn Lys Ile Gln Asp Ile Gln Leu Lys Thr

180 185 190

Ser Glu Pro Asp Phe Thr Ser Ala Asn Met Arg Asp Ser Ala Glu Gly

195 200 205

Pro Lys Glu Asp Glu Glu Lys Pro Ser Ala Ser Ala Leu Glu Gln Pro

210 215 220

Ala Thr Leu Gln Glu Val Ala Ser Gln Glu Val Pro Pro Glu Leu Ala

225 230 235 240

Thr Pro Ala Pro Ala Trp Glu Pro Gln Pro Glu Pro Asp Glu Arg Leu

245 250 255

Glu Ala Ala Ala Cys Glu Val Asn Asp Leu Gly Glu Glu Glu Glu Glu

260 265 270

Glu Glu Glu Glu Asp Glu Glu Glu Glu Glu Asp Asp Asp Asp Asp Glu

275 280 285

Leu Glu Asp Glu Gly Glu Glu Glu Ala Ser Met Pro Asn Glu Asn Ser

290 295 300

Val Lys Glu Pro Glu Ile Arg Cys Asp Glu Lys Pro Glu Asp Leu Leu

305 310 315 320

Glu Glu Pro Lys Thr Thr Ser Glu Glu Thr Leu Glu Asp Cys Ser Glu

325 330 335

Val Thr Pro Ala Met Gln Ile Pro Arg Thr Lys Glu Glu Ala Asn Gly

340 345 350

Asp Val Phe Glu Thr Phe Met Phe Pro Cys Gln His Cys Glu Arg Lys

355 360 365

Phe Thr Thr Lys Gln Gly Leu Glu Arg His Met His Ile His Ile Ser

370 375 380

Thr Val Asn His Ala Phe Lys Cys Lys Tyr Cys Gly Lys Ala Phe Gly

385 390 395 400

Thr Gln Ile Asn Arg Arg Arg His Glu Arg Arg His Glu Ala Gly Leu

405 410 415

Lys Arg Lys Pro Ser Gln Thr Leu Gln Pro Ser Glu Asp Leu Ala Asp

420 425 430

Gly Lys Ala Ser Gly Glu Asn Val Ala Ser Lys Asp Asp Ser Ser Pro

435 440 445

Pro Ser Leu Gly Pro Asp Cys Leu Ile Met Asn Ser Glu Lys Ala Ser

450 455 460

Gln Asp Thr Ile Asn Ser Ser Val Val Glu Glu Asn Gly Glu Val Lys

465 470 475 480

Glu Leu His Pro Cys Lys Tyr Cys Lys Lys Val Phe Gly Thr His Thr

485 490 495

Asn Met Arg Arg His Gln Arg Arg Val His Glu Arg His Leu Ile Pro

500 505 510

Lys Gly Val Arg Arg Lys Gly Gly Leu Glu Glu Pro Gln Pro Pro Ala

515 520 525

Glu Gln Ala Gln Ala Thr Gln Asn Val Tyr Val Pro Ser Thr Glu Pro

530 535 540

Glu Glu Glu Gly Glu Ala Asp Asp Val Tyr Ile Met Asp Ile Ser Ser

545 550 555 560

Asn Ile Ser Glu Asn Leu Asn Tyr Tyr Ile Asp Gly Lys Ile Gln Thr

565 570 575

Asn Asn Asn Thr Ser Asn Cys Asp Val Ile Glu Met Glu Ser Ala Ser

580 585 590

Ala Asp Leu Tyr Gly Ile Asn Cys Leu Leu Thr Pro Val Thr Val Glu

595 600 605

Ile Thr Gln Asn Ile Lys Thr Thr Gln Val Pro Val Thr Glu Asp Leu

610 615 620

Pro Lys Glu Pro Leu Gly Ser Thr Asn Ser Glu Ala Lys Lys Arg Arg

625 630 635 640

Thr Ala Ser Pro Pro Ala Leu Pro Lys Ile Lys Ala Glu Thr Asp Ser

645 650 655

Asp Pro Met Val Pro Ser Cys Ser Leu Ser Leu Pro Leu Ser Ile Ser

660 665 670

Thr Thr Glu Ala Val Ser Phe His Lys Glu Lys Ser Val Tyr Leu Ser

675 680 685

Ser Lys Leu Lys Gln Leu Leu Gln Thr Gln Asp Lys Leu Thr Pro Ala

690 695 700

Gly Ile Ser Ala Thr Glu Ile Ala Lys Leu Gly Pro Val Cys Val Ser

705 710 715 720

Ala Pro Ala Ser Met Leu Pro Val Thr Ser Ser Arg Phe Lys Arg Arg

725 730 735

Thr Ser Ser Pro Pro Ser Ser Pro Gln His Ser Pro Ala Leu Arg Asp

740 745 750

Phe Gly Lys Pro Ser Asp Gly Lys Ala Ala Trp Thr Asp Ala Gly Leu

755 760 765

Thr Ser Lys Lys Ser Lys Leu Glu Ser His Ser Asp Ser Pro Ala Trp

770 775 780

Ser Leu Ser Gly Arg Asp Glu Arg Glu Thr Val Ser Pro Pro Cys Phe

785 790 795 800

Asp Glu Tyr Lys Met Ser Lys Glu Trp Thr Ala Ser Ser Ala Phe Ser

805 810 815

Ser Val Cys Asn Gln Gln Pro Leu Asp Leu Ser Ser Gly Val Lys Gln

820 825 830

Lys Ala Glu Gly Thr Gly Lys Thr Pro Val Gln Trp Glu Ser Val Leu

835 840 845

Asp Leu Ser Val His Lys Lys His Cys Ser Asp Ser Glu Gly Lys Glu

850 855 860

Phe Lys Glu Ser His Ser Val Gln Pro Thr Cys Ser Ala Val Lys Lys

865 870 875 880

Arg Lys Pro Thr Thr Cys Met Leu Gln Lys Val Leu Leu Asn Glu Tyr

885 890 895

Asn Gly Ile Asp Leu Pro Val Glu Asn Pro Ala Asp Gly Thr Arg Ser

900 905 910

Pro Ser Pro Cys Lys Ser Leu Glu Ala Gln Pro Asp Pro Asp Leu Gly

915 920 925

Pro Gly Ser Gly Phe Pro Ala Pro Thr Val Glu Ser Thr Pro Asp Val

930 935 940

Cys Pro Ser Ser Pro Ala Leu Gln Thr Pro Ser Leu Ser Ser Gly Gln

945 950 955 960

Leu Pro Pro Leu Leu Ile Pro Thr Asp Pro Ser Ser Pro Pro Pro Cys

965 970 975

Pro Pro Val Leu Thr Val Ala Thr Pro Pro Pro Pro Leu Leu Pro Thr

980 985 990

Val Pro Leu Pro Ala Pro Ser Ser Ser Ala Ser Pro His Pro Cys Pro

995 1000 1005

Ser Pro Leu Ser Asn Ala Thr Ala Gln Ser Pro Leu Pro Ile Leu

1010 1015 1020

Ser Pro Thr Val Ser Pro Ser Pro Ser Pro Ile Pro Pro Val Glu

1025 1030 1035

Pro Leu Met Ser Ala Ala Ser Pro Gly Pro Pro Thr Leu Ser Ser

1040 1045 1050

Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Phe Ser Ser Ser Ser

1055 1060 1065

Ser Ser Ser Ser Pro Ser Pro Pro Pro Leu Ser Ala Ile Ser Ser

1070 1075 1080

Val Val Ser Ser Gly Asp Asn Leu Glu Ala Ser Leu Pro Met Ile

1085 1090 1095

Ser Phe Lys Gln Glu Glu Leu Glu Asn Glu Gly Leu Lys Pro Arg

1100 1105 1110

Glu Glu Pro Gln Ser Ala Ala Glu Gln Asp Val Val Val Gln Glu

1115 1120 1125

Thr Phe Asn Lys Asn Phe Val Cys Asn Val Cys Glu Ser Pro Phe

1130 1135 1140

Leu Ser Ile Lys Asp Leu Thr Lys His Leu Ser Ile His Ala Glu

1145 1150 1155

Glu Trp Pro Phe Lys Cys Glu Phe Cys Val Gln Leu Phe Lys Asp

1160 1165 1170

Lys Thr Asp Leu Ser Glu His Arg Phe Leu Leu His Gly Val Gly

1175 1180 1185

Asn Ile Phe Val Cys Ser Val Cys Lys Lys Glu Phe Ala Phe Leu

1190 1195 1200

Cys Asn Leu Gln Gln His Gln Arg Asp Leu His Pro Asp Lys Val

1205 1210 1215

Cys Thr His His Glu Phe Glu Ser Gly Thr Leu Arg Pro Gln Asn

1220 1225 1230

Phe Thr Asp Pro Ser Lys Ala His Val Glu His Met Gln Ser Leu

1235 1240 1245

Pro Glu Asp Pro Leu Glu Thr Ser Lys Glu Glu Glu Glu Leu Asn

1250 1255 1260

Asp Ser Ser Glu Glu Leu Tyr Thr Thr Ile Lys Ile Met Ala Ser

1265 1270 1275

Gly Ile Lys Thr Lys Asp Pro Asp Val Arg Leu Gly Leu Asn Gln

1280 1285 1290

His Tyr Pro Ser Phe Lys Pro Pro Pro Phe Gln Tyr His His Arg

1295 1300 1305

Asn Pro Met Gly Ile Gly Val Thr Ala Thr Asn Phe Thr Thr His

1310 1315 1320

Asn Ile Pro Gln Thr Phe Thr Thr Ala Ile Arg Cys Thr Lys Cys

1325 1330 1335

Gly Lys Gly Val Asp Asn Met Pro Glu Leu His Lys His Ile Leu

1340 1345 1350

Ala Cys Ala Ser Ala Ser Asp Lys Lys Arg Tyr Thr Pro Lys Lys

1355 1360 1365

Asn Pro Val Pro Leu Lys Gln Thr Val Gln Pro Lys Asn Gly Val

1370 1375 1380

Val Val Leu Asp Asn Ser Gly Lys Asn Ala Phe Arg Arg Met Gly

1385 1390 1395

Gln Pro Lys Arg Leu Asn Phe Ser Val Glu Leu Ser Lys Met Ser

1400 1405 1410

Ser Asn Lys Leu Lys Leu Asn Ala Leu Lys Lys Lys Asn Gln Leu

1415 1420 1425

Val Gln Lys Ala Ile Leu Gln Lys Asn Lys Ser Ala Lys Gln Lys

1430 1435 1440

Ala Asp Leu Lys Asn Ala Cys Glu Ser Ser Ser His Ile Cys Pro

1445 1450 1455

Tyr Cys Asn Arg Glu Phe Thr Tyr Ile Gly Ser Leu Asn Lys His

1460 1465 1470

Ala Ala Phe Ser Cys Pro Lys Lys Pro Leu Ser Pro Pro Lys Lys

1475 1480 1485

Lys Val Ser His Ser Ser Lys Lys Gly Gly His Ser Ser Pro Ala

1490 1495 1500

Ser Ser Asp Lys Asn Ser Asn Ser Asn His Arg Arg Arg Thr Ala

1505 1510 1515

Asp Ala Glu Ile Lys Met Gln Ser Met Gln Thr Pro Leu Gly Lys

1520 1525 1530

Thr Arg Ala Arg Ser Ser Gly Pro Thr Gln Val Pro Leu Pro Ser

1535 1540 1545

Ser Ser Phe Arg Ser Lys Gln Asn Val Lys Phe Ala Ala Ser Val

1550 1555 1560

Lys Ser Lys Lys Pro Ser Ser Ser Ser Leu Arg Asn Ser Ser Pro

1565 1570 1575

Ile Arg Met Ala Lys Ile Thr His Val Glu Gly Lys Lys Pro Lys

1580 1585 1590

Ala Val Ala Lys Asn His Ser Ala Gln Leu Ser Ser Lys Thr Ser

1595 1600 1605

Arg Ser Leu His Val Arg Val Gln Lys Ser Lys Ala Val Leu Gln

1610 1615 1620

Ser Lys Ser Thr Leu Ala Ser Lys Lys Arg Thr Asp Arg Phe Asn

1625 1630 1635

Ile Lys Ser Arg Glu Arg Ser Gly Gly Pro Val Thr Arg Ser Leu

1640 1645 1650

Gln Leu Ala Ala Ala Ala Asp Leu Ser Glu Asn Lys Arg Glu Asp

1655 1660 1665

Gly Ser Ala Lys Gln Glu Leu Lys Asp Phe Ser Tyr Ser Leu Arg

1670 1675 1680

Leu Ala Ser Arg Cys Ser Pro Pro Ala Ala Pro Tyr Ile Thr Arg

1685 1690 1695

Gln Tyr Arg Lys Val Lys Ala Pro Ala Ala Ala Gln Phe Gln Gly

1700 1705 1710

Pro Phe Phe Lys Glu

1715

<210> 13

<211> 230

<212> PRT

<213> Intelligent people

<400> 13

Met Glu Gly Gln Arg Trp Leu Pro Leu Glu Ala Asn Pro Glu Val Thr

1 5 10 15

Asn Gln Phe Leu Lys Gln Leu Gly Leu His Pro Asn Trp Gln Phe Val

20 25 30

Asp Val Tyr Gly Met Asp Pro Glu Leu Leu Ser Met Val Pro Arg Pro

35 40 45

Val Cys Ala Val Leu Leu Leu Phe Pro Ile Thr Glu Lys Tyr Glu Val

50 55 60

Phe Arg Thr Glu Glu Glu Glu Lys Ile Lys Ser Gln Gly Gln Asp Val

65 70 75 80

Thr Ser Ser Val Tyr Phe Met Lys Gln Thr Ile Ser Asn Ala Cys Gly

85 90 95

Thr Ile Gly Leu Ile His Ala Ile Ala Asn Asn Lys Asp Lys Met His

100 105 110

Phe Glu Ser Gly Ser Thr Leu Lys Lys Phe Leu Glu Glu Ser Val Ser

115 120 125

Met Ser Pro Glu Glu Arg Ala Arg Tyr Leu Glu Asn Tyr Asp Ala Ile

130 135 140

Arg Val Thr His Glu Thr Ser Ala His Glu Gly Gln Thr Glu Ala Pro

145 150 155 160

Ser Ile Asp Glu Lys Val Asp Leu His Phe Ile Ala Leu Val His Val

165 170 175

Asp Gly His Leu Tyr Glu Leu Asp Gly Arg Lys Pro Phe Pro Ile Asn

180 185 190

His Gly Glu Thr Ser Asp Glu Thr Leu Leu Glu Asp Ala Ile Glu Val

195 200 205

Cys Lys Lys Phe Met Glu Arg Asp Pro Asp Glu Leu Arg Phe Asn Ala

210 215 220

Ile Ala Leu Ser Ala Ala

225 230

<210> 14

<211> 244

<212> PRT

<213> Intelligent people

<400> 14

Met Asn Gly Pro Ala Asp Gly Glu Val Asp Tyr Lys Lys Lys Tyr Arg

1 5 10 15

Asn Leu Lys Arg Lys Leu Lys Phe Leu Ile Tyr Glu His Glu Cys Phe

20 25 30

Gln Glu Glu Leu Arg Lys Ala Gln Arg Lys Leu Leu Lys Val Ser Arg

35 40 45

Asp Lys Ser Phe Leu Leu Asp Arg Leu Leu Gln Tyr Glu Asn Val Asp

50 55 60

Glu Asp Ser Ser Asp Ser Asp Ala Thr Ala Ser Ser Asp Asn Ser Glu

65 70 75 80

Thr Glu Gly Thr Pro Lys Leu Ser Asp Thr Pro Ala Pro Lys Arg Lys

85 90 95

Arg Ser Pro Pro Leu Gly Gly Ala Pro Ser Pro Ser Ser Leu Ser Leu

100 105 110

Pro Pro Ser Thr Gly Phe Pro Leu Gln Ala Ser Gly Val Pro Ser Pro

115 120 125

Tyr Leu Ser Ser Leu Ala Ser Ser Arg Tyr Pro Pro Phe Pro Ser Asp

130 135 140

Tyr Leu Ala Leu Gln Leu Pro Glu Pro Ser Pro Leu Arg Pro Lys Arg

145 150 155 160

Glu Lys Arg Pro Arg Leu Pro Arg Lys Leu Lys Met Ala Val Gly Pro

165 170 175

Pro Asp Cys Pro Val Gly Gly Pro Leu Thr Phe Pro Gly Arg Gly Ser

180 185 190

Gly Ala Gly Val Gly Thr Thr Leu Thr Pro Leu Pro Pro Pro Lys Met

195 200 205

Pro Pro Pro Thr Ile Leu Ser Thr Val Pro Arg Gln Met Phe Ser Asp

210 215 220

Ala Gly Ser Gly Asp Asp Ala Leu Asp Gly Asp Asp Asp Leu Val Ile

225 230 235 240

Asp Ile Pro Glu

<210> 15

<211> 271

<212> PRT

<213> Intelligent people

<400> 15

Met Asn Leu Leu Gly Ser Arg Arg Val Phe Ser Lys Lys Cys Arg Leu

1 5 10 15

Val Lys Phe Ser Met Val Ala Leu Val Ser Ala Thr Met Ala Val Thr

20 25 30

Thr Val Thr Leu Glu Asn Thr Ala Leu Ala Arg Gln Thr Gln Val Ser

35 40 45

Asn Asp Val Val Leu Asn Asp Gly Ala Ser Lys Tyr Leu Asn Glu Ala

50 55 60

Leu Ala Trp Thr Phe Asn Asp Ser Pro Asn Tyr Tyr Lys Thr Leu Gly

65 70 75 80

Thr Ser Gln Ile Thr Pro Ala Leu Phe Pro Lys Ala Gly Asp Ile Leu

85 90 95

Tyr Ser Lys Leu Asp Glu Leu Gly Arg Thr Arg Thr Ala Arg Gly Thr

100 105 110

Leu Thr Tyr Ala Asn Val Glu Gly Ser Tyr Gly Val Arg Gln Ser Phe

115 120 125

Gly Lys Asn Gln Asn Pro Ala Gly Trp Thr Gly Asn Pro Asn His Val

130 135 140

Lys Tyr Lys Ile Glu Trp Leu Asn Gly Leu Ser Tyr Val Gly Asp Phe

145 150 155 160

Trp Asn Arg Ser His Leu Ile Ala Asp Ser Leu Gly Gly Asp Ala Leu

165 170 175

Arg Val Asn Ala Val Thr Gly Thr Arg Thr Gln Asn Val Gly Gly Arg

180 185 190

Asp Gln Lys Gly Gly Met Arg Tyr Thr Glu Gln Arg Ala Gln Glu Trp

195 200 205

Leu Glu Ala Asn Arg Asp Gly Tyr Leu Tyr Tyr Glu Val Ala Pro Ile

210 215 220

Tyr Asn Ala Asp Glu Leu Ile Pro Arg Ala Val Val Val Ser Met Gln

225 230 235 240

Ser Ser Asp Asn Thr Ile Asn Glu Lys Val Leu Val Tyr Asn Thr Ala

245 250 255

Asn Gly Tyr Thr Ile Asn Tyr His Asn Gly Thr Pro Thr Gln Lys

260 265 270

Claims (43)

1. A composition for expanding lymphocytes comprising interleukin 2 (IL-2), interleukin 15 (IL-15) and interleukin 21 (IL-21), wherein the composition is In liquid form, the liquid composition contains IL-2 at a concentration of 500 to 2000 U/ml, IL-15 at a concentration of 0.1 to 100 ng/ml, and wherein IL-21 is at a concentration of 0.1 to 100 ng/ml. 2. The composition of claim 1, wherein the concentration of IL-2 in the liquid composition is 800 to 1200 U/ml. 3. The composition of claim 1 or 2, wherein the concentration of IL-15 is 5 to 20 ng/ml. 4. The composition of claim 1 or 2, wherein the concentration of IL-21 is 5 to 20 ng/ml. 5. A method for preparing a clinically relevant lymphocyte population, comprising the following steps: - obtaining a body sample comprising at least one lymphocyte from a mammal, said body sample being selected from a tissue sample and a body fluid sample, - culturing said body sample in vitro in a composition comprising IL-2, IL-15 and IL-21 to expand and/or stimulate lymphocytes in said sample, wherein said composition is in liquid form, said The concentration of IL-2 in the liquid composition is 500 to 2000 U/ml, the concentration of IL-15 is 0.1 to 100 ng/ml, the concentration of IL-21 is 0.1 to 100 ng/ml, - and to determine the presence of clinically relevant lymphocytes in cultured samples; wherein the in vitro culture includes a first expansion step including incubation in a medium comprising IL-2, IL-15 and IL-21 until lymphocytes become detectable, and wherein the The in vitro culturing includes a second expansion step including incubation in a medium comprising, in addition to IL-2, IL-15 and IL-21, feeder cells and/or antibodies against CD3. 6. The method of claim 5, wherein the body sample is a tissue sample, and in step a) cells in the tissue sample are isolated. 7. The method of claim 5, wherein the clinically relevant lymphocytes are selected from the group consisting of tumor-reactive lymphocytes, pathogen-reactive lymphocytes, and autoimmune-reactive lymphocytes. 8. The method according to claim 5 or 6, wherein the body sample is selected from peripheral blood, umbilical cord blood, bone marrow, lymph node, liver, pleural effusion, pleural cavity, abdominal cavity, synovial fluid, peritoneum, retroperitoneal space, thymus and tumors. 9. The method of claim 7, wherein the body sample is selected from peripheral blood. 10. The method of claim 5, wherein the mammal is selected from the group consisting of mammals suffering from neoplastic diseases, mammals at risk of developing neoplastic diseases, mammals suffering from infectious diseases, mammals suffering from infectious diseases Mammals at risk for disease, mammals with autoimmune disease, mammals at risk for autoimmune disease. 11. The method of claim 10, wherein the mammal is a human. 12. The method of claim 5, wherein the incubation time of the first amplification step is 4 to 10 days. 13. The method of claim 5, wherein the ratio of feeder cells to lymphocytes is 1 :2 to 1 :50. 14. The method of claim 5, wherein the first expansion step comprises adding IL-2, IL-15 and IL-21 to the cell culture at the same time point. 15. The method of claim 5, wherein the first expansion step comprises separately adding at least one of IL-2, IL-15 and IL-21 to the cell culture at different time points. 16. The method of claim 13, wherein IL-21 is added first, followed by IL-15, followed by IL-2. 17. The method of claim 14, wherein IL-15 is added first, followed by IL-21, followed by IL-2. 18. The method of claim 5, wherein the clinically relevant lymphocyte population is one of monoclonal, oligoclonal, or polyclonal. 19. The method of claim 5, wherein the medium of the first expansion step and/or the second expansion step comprises at least one expansion antigen. 20. The method of claim 19, wherein the amplified antigen is a fragment of TAA comprising a peptide of at least 8 contiguous amino acids of an amino acid sequence that is at least 80% identical to the following amino acid sequence: SEQ ID NO: 4. SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 10 ID NO:13 or SEQ ID NO:14. 21. The method of claim 5, wherein the in vitro culturing further comprises a reconcentration step comprising incubating in a medium comprising reconcentrated cells. 22. The method of claim 21, wherein the time of the refocusing step is from 1 to 6 days. 23. The method of claim 21 or 22, wherein the ratio of refocused cells to lymphocytes is from 1:1 to 1:100. 24. The method of claim 5, wherein the culturing comprises adding a promoter compound to promote expansion of a specific subset of lymphocytes. 25. The method of claim 5, further comprising isolating a clinically relevant lymphocyte population from the expanded cell culture. 26. The method of claim 5, wherein detecting the presence of clinically relevant lymphocytes comprises using an evaluation antigen. 27. The method of claim 26, wherein the evaluation antigen is presented to the cultured sample in a form selected from the group consisting of cells derived from the same mammal as the cultured sample (autologous cells); at least part of the gene Matched allogeneic cells; or cells expressing clinically relevant antigens as transgenes. 28. The method of claim 26, wherein the cells are tumor cells. 29. The method of claim 27, wherein detecting the presence of clinically reactive lymphocytes comprises contacting the lymphocytes with at least one clinically relevant antigen, and measuring cytokine production, cell proliferation, cytotoxicity, signaling, and /or changes in either of intracellular phosphorylation. 30. Use of the composition according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment or prevention of infectious diseases, autoimmune diseases or tumor diseases. 31. The use of claim 29, wherein the use comprises the use of the method of any one of claims 5 to 26 to generate a clinically relevant lymphocyte population. 32. A kit for immunotherapy, wherein the kit comprises IL-2, IL-15 and IL-21, and comprises at least one of the following: a component that stimulates TCR, a costimulatory molecule, a feeder cell, and A peptide comprising the sequence of at least one clinically relevant antigen, wherein the kit comprises IL-2, IL-15 and IL-21 in a liquid composition at a concentration of IL-2 500 to 2000 U/ml, IL-15 at a concentration of 0.1 to 100 ng/ml, and IL-21 at a concentration of 0.1 to 100 ng/ml. 33. The clinically relevant lymphocytes obtained by the method of any one of claims 5 to 29, wherein the clinically relevant lymphocytes are selected from B-cells, NK cells and T cells, wherein the T cells are selected from helper T cells ( ^{TH- cells or CD4+} _-T cells); cytotoxic T cells ( _TC- cells or CD8 ⁺ -T cells); memory T cells, stem-like memory T cells ( _TSCM ) or peripheral memory cells ( T _PM- cells); γ-δ T cells (γδ-T cells); NK-T cells; mucosa-associated invariant T cells (MAIT); double negative T cells (CD3 ⁺ CD4 ⁻ CD8 ⁻ T cells). 34. The clinically relevant lymphocyte of claim 33, wherein the lymphocyte is selected from the group consisting of - lymphocytes expressing molecules that facilitate entry into tissues; and - Lymphocytes enriched in any of the following: markers of long-term memory, markers of antigen-specific immune response activation, markers of cytolytic immune cell responses. 35. Lymphocytes obtained by the method of any one of claims 5 to 29, - expression of molecules and cytokines that promote the formation of lymphocyte assemblies for medical applications, including T cell precursors, T _CM and/or T _PM ; - expression of molecules and cytokines that promote the expansion of clinically relevant lymphocytes; - production of cytokines selected from the group consisting of IFNγ, TNFα, IL-2, IL-17 and any combination thereof; - are CD3 ⁺ CD4 ^- CD8 ^- T cells. 36. A clinically relevant lymphocyte population obtained by the method of any one of claims 5 to 29. 37. The clinically relevant lymphocyte population obtained or produced by the method of any one of claims 5 to 29 is used in the preparation of a medicament for immunotherapy for treating or preventing infectious diseases, tumor diseases or autoimmune diseases of mammals for use comprising administering said clinically relevant lymphocyte population to said mammal, wherein said body sample is obtained from said mammal. 38. The use according to claim 37, wherein the neoplastic disease is selected from glioblastoma and pancreatic cancer. 39. The use of claim 37, wherein the clinically relevant lymphocyte population - causing regression of cancer cells in said mammal; - interfere with the development of precancerous lesions into malignant lesions; - Causes rapid senescence of tumor cells or precancerous cells; - causes the removal of autoantigen-positive cells; - kill pathogens, cause pathogen growth arrest or contain pathogens; - Interfere with cancer stem cells; and/or - Induces growth arrest of cancer cells or cells expressing self-antigens. 40. A lymphocyte population obtained by the method of any one of claims 5 to 29, comprising a clinically relevant lymphocyte population. 41. The lymphocyte population of claim 40, characterized by having one or more of the following characteristics: - The percentage of T _regs is less than 5% based on the total number of T cells; - The percentage of _{TH1 -cells} is at least 50% based on the total number of _TH -cells; - The percentage of CXCR3 ⁺ T cells is at least 50% based on the total number of CD8 ⁺ T cells; - The percentage of 4-1BB+ T cells is at least 1% based on the total number of T cells; - The percentage of CD117+ T cells is at least 1% based on the total number of T cells; - The percentage of CD3+CD4-CD8- cells is at least 1% based on the total number of T cells; and - The percentage of γδ T cells is at least 1% based on the total number of T cells. 42. The lymphocyte population of claim 40, characterized by having one or more of the following characteristics: - The percentage of precursor T cells (CD45RA+CCR7+) is at least 1% based on the total number of T cells; - Percentage of central memory T cells (CD45RA-CCR7+) of at least 2% based on the total number of T cells; - Percentage of peripheral memory T cells (CD45RA-CCR7-) of at least 2% based on the total number of T cells; and - The percentage of effector T cells (CD45RA+CCR7-) is at least 1% based on the total number of T cells. 43. The lymphocyte population of claim 40, characterized by having one or more of the following characteristics: - a percentage of clinically relevant T cells of at least 0.1%, based on the total number of T cells, as determined by multimeric soluble MHC-peptide complexes or intracellular cytokine production; - The value of intracellular cytokine production after antigen stimulation is at least 2 times the standard deviation in the absence of antigen stimulation; - The value of CD107a induction after antigen stimulation is at least 2 times the standard deviation of the absence of antigen stimulation.

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